NF- kappa B activating gene

ABSTRACT

Provided are proteins having NF-κ B activity, which are used for diagnosing, treating or preventing diseases associated with the excessive activation or inhibition of NF-κ B. Using plasmid pNFκ B-Luc, cDNA encoding a protein capable of activating NF-κ B has been cloned from a cDNA library constructed from human lung fibroblasts, and the DNA sequence and the deduced amino acid sequence determined. The protein, the DNA encoding the protein, a recombinant vector containing the DNA, and a transformant containing the recombinant vector are useful for screening a substance inhibiting or promoting NF-κ B activation.

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application is a continuation-in-part of application Ser.No. 10/042,211 filed on Jan. 11, 2002, which is a continuation ofPCT/JP01/11389 having an international filing date of Dec. 25, 2001,which designated the United States of America. This application andapplication Ser. No. 10/042,211 are also each a continuation-in-part ofapplication Ser. No. 10/024,298 filed on Dec. 21, 2001, which claimspriority under 35 U.S.C. § 119 (e) on U.S. Provisional Application No.60/258,315 filed on Dec. 28, 2000; No. 60/278,640 filed on Mar. 26,2001; and No. 60/314,385 filed on Aug. 24, 2001. This application alsoclaims priority under 35 U.S.C. § 119 (a) on Japanese Application Nos.402288/2000 filed Dec. 28, 2000; 088912/2001 filed on Mar. 26, 2001; and254018/2001 filed on Aug. 24, 2001. The entire contents of all of theabove—identified applications is hereby incorporated by reference.

TECHNICAL FIELD

[0002] The present invention relates to a protein capable of activatingNF-κ B, a DNA sequence encoding the protein, a method for obtaining theDNA, a recombinant vector containing the DNA, a transformant containingthe recombinant vector, and an antibody which specifically reacts withthe protein. The present invention also relates to use of the protein,DNA molecule or antibody of the invention in the diagnosis, treatment orprevention of diseases associated with the excessive activation orinhibition of NF-κ B.

[0003] The present invention also relates to a method for screening asubstance capable of inhibiting or promoting NF-κ B activation by usingthe protein, DNA, recombinant vector and transformant.

BACKGROUND ART

[0004] The transcription factor NF-κ B (nuclear factor kappa B) plays animportant role in the transcriptional regulation of various genesinvolved in inflammation and immunological reactions. NF-κ B is a homo-or heterodimer protein which belongs to the Rel family. In unstimulatedconditions, NF-κ B normally resides in the cytoplasm as an inactive formby forming a complex with an I κ B inhibitory protein (Inhibitor of NF-κB) to mask the nuclear transport signal of NF-κ B.

[0005] When cytokines such as interleukin (IL)-1 and tumor necrosisfactor (TNF)-α stimulate cells, I κ B is phosphorylated by IKK (I κ Bkinase) and degraded by the 26S proteasome through ubiquitination. Thereleased NF-κ B moves to the nucleus, where it binds to the DNA sequencecalled the NF-κ B binding sequence and induces the transcription of thegene, which is under control of NF-κ B is believed to regulate theexpression of genes such as those for immunoglobulins, inflammatorycytokines (e.g., IL-1 and TNF-α), interferons and cell adhesion factors.NF-κ B is involved in inflammation and immune responses through theexpression induction of these genes.

[0006] The inhibition of the function or activation of NF-κ B mayinhibit the expression of many factors (proteins) involved ininflammatory or immunological diseases or other diseases such as tumorproliferation. Thus, NF-κ B is a promising target for medicamentsagainst diseases caused or characterized by autoimmunity or inflammation[see e.g., Clinical Chemistry 45, 7-17 (1999); J Clin. Pharmacol. 38,981-993 (1998); Gut 43, 856-860 (1998); The New England Journal ofMedicine 366, 1066-1071 (1997); TiPS 46-50 (1997); The FASEB Journal 9,899-909 (1995); Nature 395, 225-226 (1998); Science 278, 818-819 (1997);Cell 91, 299-302 (1997)].

[0007] Extracellular information is converted into a certain signal,which passes through the cell membrane and goes through the cytoplasm tothe nucleus, where it regulates the expression of the target gene andcauses cell responses. Therefore the elucidation of the mechanism ofintracellular signal transduction from extracellular stimuli to NF-κ Bactivation is of very important significance, because it provides veryimportant means of developing new medicaments or therapies againstautoimmune diseases and diseases exhibiting inflammatory symptoms.

[0008] It is believed that the signal transduction pathway from certaincell stimulation to NF-κ B activation includes many steps mediated byvarious transmitters such as protein kinases. Therefore it is desirablefor more efficient drug discovery to identify the transmitters whichplay a key role in the pathway, and to focus research on thetransmitters to establish a new drug-screening method. Some signalingmolecules involved in NF-κ B activation have been identified [e.g., IKK,ubiquitination enzymes and the 26S proteasome described above, as wellas TNF receptor associated factor 2 (TRAF2) and NF-κ B inducing kinase(NIK)]. However, most of the mechanism of NF-κ B activation remainsunknown, and it has been desired new signaling molecules to beidentified and further the NF-κ B activation mechanism to be elucidated.

DISCLOSURE OF THE INVENTION

[0009] The object of the present invention is to identify a new gene andprotein capable of directly, or indirectly, activating NF-κ B, and toprovide a method of use of them in medicaments, diagnostics and therapy.That is, the present invention provides a new protein capable ofactivating NF-κ B, a DNA sequence encoding the protein, a recombinantvector containing the DNA, a transformant containing the recombinantvector, a process for producing the protein, an antibody directedagainst the protein or a peptide fragment thereof, and a process forproducing the antibody.

[0010] The present invention also provides a method for screening asubstance capable of inhibiting or promoting NF-κ B activation, a kitfor the screening, a substance capable of inhibiting or promoting NF-κ Bactivation obtainable by the screening method or the screening kit, aprocess for producing the substance, a pharmaceutical compositioncontaining a substance capable of inhibiting or promoting NF-κ Bactivation, etc.

[0011] Recently, random analysis of cDNA molecules has been intensivelycarried out to analyze various genes, which are expressed in vivo. ThecDNA fragments thus obtained have been entered for databases andpublished as ESTs (Expressed Sequence Tags, e.g.,http//www.ncbi.nlm.nih.gov/dbEST). However, ESTs are merely sequenceinformation, and it is difficult to predict their functions. ESTs arealso arranged in UniGene (http//www.ncbi.nlm.nih.gov/UniGene), and about92,000 clusters have been registered until now. However, most of theseESTs have their 5′ end nucleotide sequences deleted, and contain notranslation initiation site. Therefore it is unlikely that such analysiswill directly lead to gene functional analysis such as the analysis ofprotein functions on the assumption of the determination of mRNA codingregions and the understanding of gene expression control by the analysisof promoters.

[0012] On the other hand, one method to elucidate functions of geneproducts (i.e., proteins) is transient expression cloning method usinganimal cells [see e.g., “Idenshi Kougaku Handbook (Genetic EngineeringHandbook)”, an extra issue of “Jikken Igaku (Experimental Medicine)”,YODOSHA CO., LTD.]. This method involves transfecting animal cells witha cDNA library constructed using an animal cell expression vector todirectly express a functional protein, and identifying and cloning thecDNA based on the biological activity of the protein having an effect onthe cells. This method requires no chemical information (amino acidsequences and molecular weights) regarding the target protein product asa prerequisite, and allows the identification of cDNA clones bydetecting specific biological activity of the protein expressed in thecells or culture.

[0013] For the efficient expression cloning, there is a need to devise amethod of preparing a cDNA library. Several methods have been widelyused to construct cDNA libraries [e.g., the method of Gubbler-Hoffman:Gene 25 (1983); and the method of Okayama-Berg: Mol. Cell. Biol. 2(1982)]. However, most of the cDNA molecules prepared by these methodshave their 5′ end nucleotide sequences deleted, and thus these methodsrarely produce full-length cDNA, a complete DNA copy of mRNA. This isbecause the reverse transcriptase used to prepare cDNA from mRNA doesnot necessarily have high efficiency in producing full-length cDNA.Therefore it is necessary to improve these prior art methods in order toefficiently carry out the above expression cloning.

[0014] In addition, in order to carry out the functional analysis ofgenes, it is essential to clone full-length cDNA sequences and expressproteins from them. Therefore, it has been necessary to construct cDNAlibraries containing enriched full-length cDNA for efficient expressioncloning.

[0015] The present inventors have intensively studied to solve the aboveproblems. As a result, the present inventors have succeeded inconstructing a full-length cDNA library by using the oligo-cappingmethod; establishing a gene function assay system by expression cloningusing 293-EBNA cells; and isolating a new DNA (cDNA) encoding a proteinhaving a function of activating NF-κ B by using the assay system. Thisnew DNA molecule induced NF-κ B activation by its expression in 293-EBNAcells. This result shows that this new DNA is a signal transductionmolecule involved in NF-κ B activation. Thus, the present invention hasbeen completed.

[0016] That is, the present invention relates to:

[0017] (1) A purified protein selected from the group consisting of:

[0018] (a) a protein that activates NF-κ B which consists of an aminoacid sequence represented by any one of SEQ ID NOS: 1, 3, 5, 7, 9, 11,13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47,49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83,85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115,117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143,145, 147, 149, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172,174, 176, 178, and 180; and

[0019] (b) a protein that activates NF-κ B and consists of an amino acidsequence having at least one amino acid deletion, substitution oraddition in an amino acid sequence represented by any one of SEQ ID NOS:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37,39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73,75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107,109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135,137, 139, 141, 143, 145, 147, 149, 152, 154, 156, 158, 160, 162, 164,166, 168, 170, 172, 174, 176, 178 and 180.

[0020] (2) A purified protein that activates NF-κ B and comprises anamino acid sequence having at least 50% identity to the proteinaccording to above item (1) over the entire length thereof.

[0021] (3) An isolated polynucleotide which comprises a nucleotidesequence encoding a protein selected from the group consisting of:

[0022] (a) a protein which comprises an amino acid sequence representedby any one of SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25,27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61,63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97,99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125,127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 152, 154,156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178 and 180; and

[0023] (b) a protein that activates NF-κ B and consists of an amino acidsequence having at least one amino acid deletion, substitution oraddition in an amino acid sequence represented by any one of SEQ ID NOS:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37,39, 41, 43, 45, 47,49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73,75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107,109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135,137, 139, 141, 143, 145, 147, 149, 152, 154, 156, 158, 160, 162, 164,166, 168, 170, 172, 174, 176, 178 and 180.

[0024] (4) An isolated polynucleotide comprising a polynucleotidesequence selected from the group consisting of:

[0025] (a) a polynucleotide sequence represented by any one of SEQ IDNOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36,38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72,74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106,108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134,136, 138, 140, 142, 144, 146, 148, 150, 151, 153, 155, 157, 159, 161,163, 165, 167, 169, 171, 173, 175, 177 and 179;

[0026] (b) a polynucleotide sequence encoding a protein that activatesNF-κ B and hybridizing under stringent conditions with a polynucleotidehaving a polynucleotide sequence complementary to the polynucleotidesequence of (a); and

[0027] (c) a polynucleotide sequence which encodes a protein thatactivates NF-κ B and consists of a polynucleotide sequence having atleast one nucleotide deletion, substitution or addition in apolynucleotide sequence represented by any one of SEQ ID NOS: 2, 4, 6,8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42,44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78,80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110,112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138,140, 142, 144, 146, 148, 150, 151, 153, 155, 157, 159, 161, 163, 165,167, 169, 171, 173, 175, 177 and 179.

[0028] (5) An isolated polynucleotide comprising a polynucleotidesequence selected from the group consisting of:

[0029] (a) a nucleotide sequence represented by a coding region in anyone of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28,30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64,66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100,102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128,130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 151, 153, 155,157, 159, 161, 163, 165, 167, 169, 171, 173, 175, 177 and 179;

[0030] (b) a nucleotide sequence encoding a protein that activates NF-κB and hybridizing under stringent conditions with a polynucleotidehaving a polynucleotide sequence complementary to the polynucleotidesequence of (a); and

[0031] (c) a nucleotide sequence which encodes a protein that activatesNF-κ B and consists of a nucleotide sequence having at least onenucleotide deletion, substitution or addition in a nucleotide sequencerepresented by a coding region in any one of SEQ ID NOS: 2, 4, 6, 8, 10,12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46,48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82,84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114,116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142,144, 146, 148, 150, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169,171, 173, 175, 177 and 179.

[0032] (6) An isolated polynucleotide comprising a nucleotide sequencewhich encodes a protein that activates NF-κ B and has at least 95%identity to the polynucleotide sequence according to above item (3) overthe entire length thereof.

[0033] (7) An isolated polynucleotide comprising a nucleotide sequencewhich encodes a protein that activates NF-κ B and has at least 95%identity to the polynucleotide sequence according to above item (4) or(5) over the entire length thereof.

[0034] (8) A purified protein encoded by the polynucleotide according toany one of above items (3) to (7).

[0035] (9) A recombinant vector which comprises a polynucleotideaccording to any one of above items (3) to (7).

[0036] (10) A gene thrapy agent comprising the recombinant vectoraccording to above item (9) as an active ingredient.

[0037] (11) A transformed cell which comprises the recombinant vectoraccording to above item (9).

[0038] (12) A membrane of the cell according to above item (11), whenthe protein according to above item (1) or (2) is a membrane protein.

[0039] (13) A process for producing a protein comprising,

[0040] (a) culturing a transformed cell comprising the isolatedpolynucleotide according to any one of items (3) to (7) under conditionsproviding expression of the encoded protein; and

[0041] (b) recovering the protein from the culture.

[0042] (14) A process for diagnosing a disease or a susceptibility to adisease in a subject related to expression or activity of the proteinaccording to above item (1), (2) or (8) in a subject comprising:

[0043] (a) determining the presence or absence of a mutation in thenucleotide sequence encoding said protein in the genome of said subject;and/or

[0044] (b) analyzing the amount of expression of said protein in asample derived from said subject. In the above-described method, adiagnosis of disease is preferably made when the amount of the proteinexpressed is 2-fold or higher than normal, or half or less than normal.

[0045] (15) A method for screening a compound in respect of activity toinhibit or promote NF-κ B activation, which comprises the steps of:

[0046] (a) providing a cell with a gene encoding a protein thatactivates NF-κ B, and a component that provides a detectable signalassociated with activation of NF-κ B;

[0047] (b) culturing a transformed cell under conditions, which permitthe expression of the gene in the transformed cell;

[0048] (c) contacting the transformed cell with one or more compounds;

[0049] (d) measuring the detectable signal; and

[0050] (e) isolating or identifying an activator compound and/or aninhibitor compound by measuring the detectable signal.

[0051] Further, it is preferable to isolate or identify as an activatorcompound, a compound that increases said detectable signal 2-fold orhigher than normal, and to isolate or identify as an inhibitor compound,a compound that decreases said detectable signal half or less thannormal.

[0052] (16) A process for producing a pharmaceutical composition, whichcomprises the steps of:

[0053] (a) providing a cell with a gene encoding a protein thatactivates NF-κ B, and a component capable of providing a detectablesignal;

[0054] (b) culturing a transformed cell under conditions, which permitthe expression of the gene in the transformed cell;

[0055] (c) contacting the transformed cell with one or more candidatecompounds;

[0056] (d) measuring the detectable signal;

[0057] (e) isolating or identifying an activator compound and/or aninhibitor compound by measuring the detectable signal; and

[0058] (f) optimizing the isolated or identified compound as apharmaceutical composition.

[0059] Further, it is preferable to isolate or identify as an activatorcompound, a compound that increases said detectable signal 2-fold orhigher than normal, and to isolate or identify as an inhibitor compound,a compound that decreases said detectable signal half or less thannormal.

[0060] (17) A kit for screening a compound in respect of activity toinhibit or promote NF-κ B activation, which comprises:

[0061] (a) a cell comprising a gene encoding a protein that activatesNF-κ B, and a component that provides a detectable signal uponactivation of NF-κ B; and

[0062] (b) reagents for measuring the detectable signal.

[0063] (18) A monoclonal or polyclonal antibody or a fragment thereofthat specifically binds to the protein according to above item (1), (2)or (8).

[0064] (19) The monoclonal or polyclonal antibody or a fragment thereofaccording to above item (18) that inhibits the action of activating NF-κB of the protein according to above item (1), (2) or (8).

[0065] (20) A process for producing a monoclonal or polyclonal antibodyaccording to above item that specifically binds to the protein of aboveitem (1), (2) or (8), which comprises administering the proteinaccording to above item (1), (2) or (8) as an antigen or epitope-bearingfragments to a non-human animal.

[0066] (21) An antisense oligonucleotide complementary to thepolynucleotide according to any one of above items (3) to (7),whichprevents NF-κ B activator protein expression.

[0067] (22) A ribozyme which inhibits NF-κ B activation by cleavage ofRNA that encodes the protein of above item (1), (2) or (8).

[0068] (23) A double stranded nucleic acid having a nucleotide sequencecorresponding to a part of the nucleotide sequence of the isolatedpolynucleotide according to any one of above items 3-7, which inhibitthe expression of the protein that activates NF-κ B.

[0069] (24) The double stranded nucleic acid according to above item 23,wherein the nucleic acid has a nucleotide sequence corresponding to apart of the nucleotide sequence represented by SEQ ID NO: 88, andinhibits the expression of the protein having the amino acid sequencerepresented by SEQ ID NO: 87.

[0070] (25) A double stranded nucleic acid obtained by annealing of anyone of the following oligonucleotide pairs (a)-(f): (a)5′-GUCCAGGAUAUCAUGAGUCN_(n)-3′ (SEQ ID NO:213)3′-N_(n)CAGGUCCUAUAGUACUCAG-5′ (SEQ ID NO:214) (b)5′-GAAGUCUGAAGAUCUAUCCN_(n)-3′ (SEQ ID NO:215)3′-N_(n)CUUCAGACUUCUAGAUAGG-5′ (SEQ ID NO:216) (c)5′-GCUGAAGAAGAGGUGUUCCN_(n)-3′ (SEQ ID NO:217)3′-N_(n)CGACUUCUUCUCCACAAGG-5′ (SEQ ID NO:218) (d)5′-GAUGACACAGAUGAAGCCCN_(n)-3′ (SEQ ID NO:219)3′-N_(n)CUACUGUGUCUACUUCGGG-5′ (SEQ ID NO:220) (e)5′-GCCCUCAGAGUCCAGAAUCN_(n)-3′ (SEQ ID NO:221)3′-N_(n)CGGGAGUCUCAGGUCUUAG-5′ (SEQ ID NO:222) (f)5′-GAUGACUUUGGUAUCAAACN_(n)-3′ (SEQ ID NO:223)3′-N_(n)CUACUGAAACCAUAGUUUG-5′ (SEQ ID NO:224)

[0071] wherein N represents any one of G, A, T, C, and U, and n is 1 to4.

[0072] It should be noted that “N_(n)” is not included in each sequenceof SEQ ID NOs: 213-224 in the Sequence Listing.

[0073] (26) The double stranded nucleic acid according to above item 25,wherein Nn is TT or UU.

[0074] (27) A double stranded nucleic acid having one or more mutationsin the sense strand of the double strand nucleic acid according to aboveitem (26).

[0075] (28) A double stranded nucleic acid comprising the doublestranded nucleic acid according to any one of above items (25) to (27)as a part, which inhibits the expression of the protein having the aminoacid sequence represented by SEQ ID NO: 87.

[0076] (29) An expression vector capable of expressing the doublestranded nucleic acid according to above item (25), wherein Nn is UU orUUU.

[0077] (30) A method for treating a disease, which comprisesadministering to a subject an amount of compound screened by the processaccording to above item (15), and/or a monoclonal or polyclonal antibodyor a fragment thereof according to above item (18) or (19), and/or anantisense oligonucleotide according to above item (21) and/or a ribozymeaccording to above item (22) and/or a double stranded nucleic acidaccording to any one of above items (23)-(28) and/or the expressionvector according to above item (29) effective to treat a diseaseselected from the group consisting of inflammation, autoimmune diseases,infectious disease, cancers and bone diseases.

[0078] (31) A method for treating a disease, which comprisesadministering to a subject an amount of a compound screened by theprocess according to above item (15), and/or a monoclonal or polyclonalantibody or a fragment thereof according to above item (18) or (19),and/or an antisense oligonucleotide according to above item (21), and/ora ribozyme according to above item (22) effective to treat a diseaseselected from the group consisting of inflammation, autoimmune diseases,infectious diseases, cancers and bone diseases.

[0079] (32) A pharmaceutical composition produced according to theprocess of item (16) as an inhibitor or promoter of NF-κ B activation.

[0080] (33) A pharmaceutical composition according to item (32) for thetreatment of inflammation, autoimmune diseases, cancers, infectiousdiseases, bone diseases, AIDS, neurodegenerative diseases, or ischemicdisorders.

[0081] (34) A method of treating inflammation, autoimmune diseases,cancers, infectious diseases, bone diseases, AIDS, neurodegenerativediseases, or ischemic disorders, which comprises administering apharmaceutical composition produced according to the process of aboveitem (16) to a patient suffering from a disease associated with NF-κ B.

[0082] (35) A pharmaceutical composition which comprises a monoclonal orpolyclonal antibody or a fragment thereof according to item (18) or (19)as an active ingredient.

[0083] (36) A pharmaceutical composition which comprises an antisenseoligonucleotide according to item (21) as an active ingredient.

[0084] (37) A pharmaceutical composition which comprises a ribozymeaccording to above item (22) as an active ingredient.

[0085] (38) A pharmaceutical composition or a gene therapy agent whichcomprises a double stranded nucleic acid according to any one of aboveitems (23) to (28) and/or an expression vextor according to above item(29) as an active ingredient.

[0086] (39) An expression inhibiting agent for a protein having anaction of activating NF-κ B, which comprises a double stranded nucleicacid according to any one of above items (23) to (28) and/or anexpression vextor according to above item (29) as an active ingredient.

[0087] (40) The pharmaceutical composition according to item (35) or(36), wherein the target disease is selected from the group consistingof inflammation, autoimmune diseases, infectious diseases, cancers, bonediseases, AIDS, neurodegenerative diseases and ischemic disorders.

[0088] (41) A method for obtaining a novel gene having a function, whichcomprises at least the following steps:

[0089] (a) constructing a full-length cDNA library by the oligo-cappingmethod;

[0090] (b) cotransfecting the full-length cDNA and a plasmid containinga factor emitting a signal indicative of the presence of a proteinhaving the function into cells; and

[0091] (c) selecting a plasmid emitting the signal.

[0092] It should be noted that a novel gene having a function accordingto the present invention refers to, for example, a nucleic acid moleculeencoding a protein having biological function.

[0093] (42) A computer-readable medium on which a sequence data set hasbeen stored, said sequence data set comprising at least one nucleotidesequence selected from the group consisting of SEQ ID NOS: 2, 4, 6, 8,10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44,46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80,82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112,114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140,142, 144, 146, 148, 150, 151, 153, 155, 157, 159, 161, 163, 165, 167,169, 171, 173, 175, 177 and 179, and/or at least one amino acid sequenceselected from the group consisting of SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13,15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49,51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85,87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117,119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145,147, 149, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174,176, 178 and 180.

[0094] (43) A method for calculating identity to other nucleotidesequences and/or amino acid sequences, which comprises comparing data ona medium according to above item (42) with data of said other nucleotidesequences and/or amino acid sequences.

[0095] (44) An insoluble substrate to which polynucleotide comprisingall or part of the nucleotide sequences selected from the groupconsisting of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24,26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60,62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88 and 90, 92, 94,96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124,126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 151,153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173, 175, 177 and 179,are fixed.

[0096] (45) An insoluble substrate to which polypeptides comprising allor a part of the amino acid sequences selected from the group consistingof SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21,23, 25, 27, 29, 31,33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67,69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101,103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129,131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 152, 154, 156, 158,160, 162, 164, 166, 168, 170, 172, 174, 176, 178 and 180, are fixed.

[0097] The contents of the specficiations and/or drawings of JapanesePatent Applications Nos. 2000-402288, 2001-088912 and 2001-254018, andU.S. Provisional Applications Nos. 60/258,315, 60/278,640 and60/314,385, which from the bases of priority of the instant application,are incorporated herein.

BRIEF DESCRIPTION OF THE DRAWINGS

[0098]FIG. 1 is a graph showing NF-κ B reporter activity inhibition bythe proteasome inhibitor MG-132 (SEQ ID NO: 5) in Example 3, the axis ofabscissa is MG-132 concentration and the transversal axis is relativeluciferase activity where relative luciferase activity is taken as 100%under conditions of non-addition of MG-132 (0 μM). (Relative luciferaseactivity at various concentrations was divided by relative luciferaseactivity under conditions of non-addition of MG-132, and expressed as apercentage.)

[0099]FIG. 2 is a graph showing NF-κ B reporter activity inhibition bythe proteasome inhibitor MG-132 (SEQ ID NO: 9) in Example 3.

[0100]FIG. 3 is a graph showing NF-κ B reporter activity inhibition bythe proteasome inhibitor MG-132 (SEQ ID NO: 17) in Example 3.

[0101]FIG. 4 is a graph showing NF-κ B reporter activity inhibition bythe proteasome inhibitor MG-132 (SEQ ID NO: 21) in Example 3.

[0102]FIG. 5 is a graph showing NF-κ B reporter activity inhibition bythe proteasome inhibitor MG-132 (SEQ ID NO: 35) in Example 3.

[0103]FIG. 6 is a graph showing NF-κ B reporter activity inhibition bythe proteasome inhibitor MG-132 (SEQ ID NO: 37) in Example 3.

[0104]FIG. 7 is a graph showing NF-κ B reporter activity inhibition bythe proteasome inhibitor MG-132 (SEQ ID NO: 41) in Example 3.

[0105]FIG. 8 is a graph showing NF-κ B reporter activity inhibition bythe proteasome inhibitor MG-132 (SEQ ID NO: 53) in Example 3.

[0106]FIG. 9 is a graph showing NF-κ B reporter activity inhibition bythe proteasome inhibitor MG-132 (SEQ ID NO: 57) in Example 3.

[0107]FIG. 10 is a graph showing NF-κ B reporter activity inhibition bythe proteasome inhibitor MG-132 (SEQ ID NO: 63) in Example 3.

[0108]FIG. 11 is a graph showing NF-κ B reporter activity inhibition bythe proteasome inhibitor MG-132 (SEQ ID NO: 67) in Example 3.

[0109]FIG. 12 is a graph showing NF-κ B reporter activity inhibition bythe proteasome inhibitor MG-132 (SEQ ID NO: 71) in Example 3.

[0110]FIG. 13 is a graph showing NF-κ B reporter activity inhibition bythe proteasome inhibitor MG-132 (SEQ ID NO: 75) in Example 3.

[0111]FIG. 14 is a graph showing NF-κ B reporter activity inhibition bythe proteasome inhibitor MG-132 (SEQ ID NO: 81) in Example 3.

[0112]FIG. 15 is a graph showing NF-κ B reporter activity inhibition bythe proteasome inhibitor MG-132 (SEQ ID NO: 87) in Example 3.

[0113]FIG. 16 is a graph showing NF-κ B reporter activity inhibition bythe proteasome inhibitor MG-132 (SEQ ID NO: 91) in Example 3.

[0114]FIG. 17 is a graph showing NF-κ B reporter activity inhibition bythe proteasome inhibitor MG-132 (SEQ ID NO: 93) in Example 3.

[0115]FIG. 18 is a graph showing NF-κ B reporter activity inhibition bythe proteasome inhibitor MG-132 (SEQ ID NO: 97) in Example 3.

[0116]FIG. 19 is a graph showing NF-κ B reporter activity inhibition bythe proteasome inhibitor MG-132 (SEQ ID NO: 121) in Example 3.

[0117]FIG. 20 is a graph showing NF-κ B reporter activity inhibition bythe proteasome inhibitor MG-132 (SEQ ID NO: 123) in Example 3.

[0118]FIG. 21 is a graph showing NF-κ B reporter activity inhibition bythe proteasome inhibitor MG-132 (SEQ ID NO: 129) in Example 3.

[0119]FIG. 22 is a graph showing NF-κ B reporter activity inhibition bythe proteasome inhibitor MG-132 (SEQ ID NO: 154) in Example 3.

[0120]FIG. 23 is a graph showing NF-κ B reporter activity inhibition bythe proteasome inhibitor MG-132 (SEQ ID NO: 158) in Example 3.

[0121]FIG. 24 is a graph showing NF-κ B reporter activity inhibition bythe proteasome inhibitor MG-132 (SEQ ID NO: 162) in Example 3.

[0122]FIG. 25 is a graph showing NF-κ B reporter activity inhibition bythe proteasome inhibitor MG-132 (SEQ ID NO: 168) in Example 3.

[0123]FIG. 26 is a graph showing NF-κ B reporter activity inhibition bythe proteasome inhibitor MG-132 (SEQ ID NO: 170) in Example 3.

[0124]FIG. 27 is a graph showing NF-κ B reporter activity inhibition bythe proteasome inhibitor MG-132 (SEQ ID NO: 172) in Example 3.

[0125]FIG. 28 is a graph showing NF-κ B reporter activity inhibition bythe proteasome inhibitor MG-132 (SEQ ID NO: 176) in Example 3.

[0126]FIG. 29 is a graph showing NF-κ B reporter activity inhibition bythe proteasome inhibitor MG-132 (SEQ ID NO: 178) in Example 3.

BEST MODE FOR CARRYING OUT THE INVENTION

[0127] At first, in order to further clarify the basic feature of thepresent invention, the present invention is explained by following howthe present invention is completed. In order to obtain a new gene havinga function of activating NF-κ B, the following experiments were carriedout as shown in the examples. First, using the oligo-capping method, afull-length cDNA was produced from mRNA prepared from normal human lungfibroblasts (purchased from. Sanko Junyaku Co., Ltd.), and a full-lengthcDNA library was constructed in which the cDNA was inserted into thevector pME18S-FL3 (GenBank Accession AB009864). Next, the cDNA librarywas introduced into E. coli cells, and plasmid preparation was carriedout per clone. Then, the pNK κ B-Luc reporter plasmid (STRATAGENE)containing a DNA encoding luciferase under control of a promoteractivated by NF-κ B and the above full-length cDNA plasmid werecotransfected into 293-EBNA cells (Invitrogen). After 24 or 48 hours ofculture, luciferase activity was measured, and the plasmid withsignificantly increased luciferase activity compared to that of acontrol experiment (vector pME18S-FL3 is introduced into a cell in placeof a full-length cDNA) was selected (the selected plasmid showed a5-fold or more increase in luciferase activity compared to that of thecontrol experiment), and the entire nucleotide sequence of the cDNAcloned into the plasmid was determined. The protein encoded by the cDNAthus obtained shows that this protein is a signal transduction moleculeinvolved in NF-κ B activation.

[0128] The present invention is described in detail below.

[0129] In the present invention, activation of NF-κ B refers to director indirect activation of NF-κ B (including induction of NF-κ Bactivation) when a gene is introduced into a suitable cell and theprotein encoded by the gene is excessively expressed. Activation of NF-κB can be measured, for example, by an assay using an NF-κ B dependantreporter gene. In the assay, activation may be reflected by increasingthe reporter activity compared to control cells (cells into which thevector only was introduced). Increase in reporter activity is preferablyby a factor of 1.5 or more, more preferably by a factor of 2 or more,and still more preferably by a factor of 5 or more.

[0130] Reporter activity can be measured by cloning a polynucleotide(e.g. cDNA) encoding the protein to be expressed into a suitableexpression vector, co-transfecting the expression vector and an NF-κ Bdependant reporter plasmid into a suitable cell, and after culturing fora certain period, then measuring reporter activity. Suitable expressionvectors are well known to those skilled in the art, examples of whichinclude pME18S-FL3, pcDNA3.1. (Invitrogen). The reporter gene can be onewhich enables a person skilled in the art to easily detect theexpression thereof, and examples include a gene encoding luciferase,chloramphenicol acetyl transferase, or β-galactosidase. Use of a geneencoding luciferase is most preferable, and examples of an NF-κ Bdependent reporter plasmid include pNF-κ B-Luc (STRATAGENE). Suitablecells include cells which exhibit an NF-κ B activation response tostimulation by IL-1, TNF-α and the like. Examples include 293-EBNAcells. Cell culture and introduction of genes into cells (transfection)can be performed and optimized by a person skilled in the art by knowntechniques.

[0131] As a preferable method, 293-EBNA cells are inoculated on 5% FBS(Fetal Bovine Serum) containing DMEM medium (Dulbecco's Modified EagleMedium) in a 96-well cell culture plate to a final cell density of 1×10⁴cells/well, and cultured for 24 hours at 37° C. in the presence of 5%CO₂. Then, reporter plasmid pNF-κ B-Luc (STRATAGENE) and the expressionvector are cotransfected into the cells in a well using FuGENE 6(Roche). After 24 hours of culture at 37° C., NF-κ B activation is thenmeasured by measuring luciferase activity using a long term luciferaseassay system, Picagene LT2.0 (Toyo Ink Mfg). For example, luciferaseactivity can be measured using PerkinElmer's Wallac ARVOTMST 1420MULTILABEL COUNTER. The method for gene introduction by FuGENE6, andmeasurement of luciferase activity by Picagene LT2.0 can be performedrespectively according to the attached protocols. In a method of geneintroduction with a 96-well plate using FuGENE6, the amount of FuGENE6per 1 well is suitably 0.3 to 0.5 μl , preferably 0.3 μl; the amount ofpNF-κ B-Luc plasmid is suitably 50 to 100 ng, preferably 50 ng; theamount of expression vector is suitably 50-100 ng, and preferably 100ng. An ability to activate NF-κ B refers to an ability to increase thereporter activity (luciferase activity) relative to the controlexperiment (cells into which only a null vector was introduced).Increase in reporter activity is preferably by a factor of 1.5 or more,more preferably by a factor of 2 or more, and still more preferably by afactor of 5 or more.

[0132] Related to the amino acid sequences of any one of SEQ ID NOS. 1,3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39,41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75,77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109,111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137,139, 141, 143, 145, 147, 149, 152, 154, 156, 158, 160, 162, 164, 166,168, 170, 172, 174, 176, 178 and 180, the present invention provides fora protein that:

[0133] (a) comprises one of the above amino acid sequences;

[0134] (b) is a peptide having one of the above amino acid sequences;

[0135] (c) activates NF-κ B and consists of an amino acid sequencehaving at least one amino acid deletion, substitution or addition in theabove amino acid sequences;

[0136] (d) comprises an amino acid sequence, which has at least 95%identity, preferably at least 97-99% identity, to an amino acid sequenceof SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31,33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67,69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101,103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129,131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 152, 154, 156, 158,160, 162, 164, 166, 168, 170, 172, 174, 176, 178 or 180, over the entirelength thereof.

[0137] “Identity” as known in the art, is a relationship between two ormore protein sequence or two or more polynucleotide sequences, asdetermined by comparing the sequences. In the art, “identity” also meansthe degree of sequence relatedness between protein or polynucleotidesequences, as determined by the match between protein or polynucleotidesequences, as the case may be, as determined by the match betweenstrings of such sequences. “Identity” and “similarity” can be readilycalculated by known methods. Preferred methods to determine identity aredesigned to give the largest match between the sequences tested. Methodsto determine identity and similarity are codified in publicly availablecomputer programs. “Identity” can be determined by using the BLASTprogram (for example, Altschul S F, Gish W, Miller W, Myers E W, LipmanD J., J. Mol. Biol., 215: p403-410 (1990), Altschul S F, Madden T L,Schaffer A A, Zhang Z, Miller W, Lipman D J,. Nucleic Acids Res. 25:p3389-3402 (1997)). Where software such as BLAST is used, it ispreferable to use default values. The main initial conditions generallyused in a BLAST search are as follows, but are not limited to these.

[0138] An amino acid substitution matrix is a matrix numericallyrepresenting the degree of analogy of each pairing of each of the 20types of amino acid, and normally the default matrix of BLOSUM62 isused. The theory of this amino acid substitution matrix is shown inAltschul S. F., J. Mol. Biol. 219: 555-565 (1991), and applicability toDNA sequence comparison is shown on States D. J., Gish W., Altschul S.F., Methods, 3: 66-70 (1991). In this case, optimal gap cost isdetermined by experience and in the case of BLOSUM62 preferablyparameters of Existence 11, Extension 1 are used. The expected value(EXPECT) is the threshold value concerning statistical significance fora match with a database sequence, and the default value is 10.

[0139] As one example, a protein having, for example, 95% or moreidentity to the amino acid sequence of SEQ ID NO: 2 may contain in theamino acid sequence up to 5 amino acid changes per 100 amino acids ofthe amino acid sequence of SEQ ID NO: 2. In other words, a proteinhaving 95% or more amino acid sequence identity to a subject amino acidsequence, may have amino acids up to 5% of the total number of aminoacids within the subject sequence, deleted or substituted by other aminoacids, or amino acids up to 5% of the total number of amino acids withinthe subject sequence may be inserted within the subject sequence. Thesechanges within the subject sequence, may exist at the amino terminus orthe carboxy terminus of the subject sequence, or may exist at anyposition between these termini, or may form one or more groups ofchanges.

[0140] The Examples described below demonstrate that the proteinconsisting of an amino acid sequence of any one of the above SEQ ID NO:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37,39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73,75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107,109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135,137, 139, 141, 143, 145, 147, 149, 152, 154, 156, 158, 160, 162, 164,166, 168, 170, 172, 174, 176, 178 and 180, is capable of activating NF-κB.

[0141] Related to the polynucleotide sequence of any one of SEQ ID NOS:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38,40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74,76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108,110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136,138, 140, 142, 144, 146, 148, 150, 151, 153, 155, 157, 159, 161, 163,165, 167, 169, 171, 173, 175, 177 and 179, the present invention furtherprovides an isolated polynucleotide that:

[0142] (a) comprises a nucleotide sequence, which has at least 95%identity, preferably at least 97-99% identity to any one of the abovesequences;

[0143] (b) is a polynucleotide of any one of the above sequences; or

[0144] (c) has a nucleotide sequence encoding a protein which has atleast 95% identity, preferably, at least 97-99% identity, to the aminoacid sequence of any one of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17,19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53,55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89,91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119,121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147,149, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176,178 and 180.

[0145] Polynucleotides which are identical or almost identical tonucleotide sequences contained in the above nucleotide sequences may beused as hybridization probes to isolate full-length cDNA and genomicclones encoding the protein of the present invention, or cDNA or genomicclones of other genes that have a high sequence similarity to the abovesequences, or genomic clones, or may be used as primers for nucleic acidamplification reactions. Typically, these nucleotide sequences are 70%identical, preferably 80% identical, more preferably 90% identical, mostpreferably 95% identical to the above sequences. The probes or primerswill generally comprises at least 15 nucleotides, preferably 30nucleotides and may have 50 nucleotides. Particularly preferred probeswill have between 30 and 50 nucleotides. Particularly preferred primershave between 20 and 25 nucleotides.

[0146] The polynucleotide of the present invention may be either in theform of a DNA such as cDNA, a genomic DNA obtained by cloning orsynthetically produced, or may be in the form of RNA such as mRNA. Thepolynucleotide may be single-stranded or double-stranded. Thedouble-stranded polynucleotides may be double-stranded DNA,double-stranded RNA or DNA:RNA hybrid. The single-strandedpolynucleotide may be sense strand also known as coding strand orantisense strand also known as non-coding strand.

[0147] Those skilled in the art can prepare a protein having the sameNF-κ B activating activity as the protein having an amino acid sequenceof any one of SEQ ID NO:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25,27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61,63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97,99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125,127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 152, 154,156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178 and 180, bymeans of appropriate substitution of an amino acid in the protein usingknown methods. One such method involves using conventional mutagenesisprocedures for the DNA encoding the protein. Another method is, forexample, site-directed mutagenesis (e.g., Mutan-Super Express Km Kitfrom Takara Shuzo Co., Ltd.). Mutations of amino acids in proteins mayalso occur in nature. Thus, the present invention also includes amutated protein which is capable of activating NF-κ B and which has atleast one amino acid deletion, substitution or addition relative to theprotein of any one of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21,23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57,59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93,95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123,125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 152,154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178 and 180,and the DNA encoding the protein. The number of mutations is preferablyup to 10, more preferably up to 5, most preferably up to 3.

[0148] The substitutions of amino acids are preferably conservativesubstitutions, specific examples of which are substitutions within thefollowing groups: (glycine, alanine), (valine, isoleucine, leucine),(aspartic acid, glutamic acid), (asparagine, glutamine), (serine,threonine), (lysine, arginine) and (phenylalanine, tyrosine).

[0149] Based on the nucleotide sequences (e.g., SEQ ID NO: 2) encoding aprotein consisting of an amino acid sequence of any one of SEQ ID NO: 1,3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39,41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75,77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109,111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137,139, 141, 143, 145, 147, 149, 152, 154, 156, 158, 160, 162, 164, 166,168, 170, 172, 174, 176, 178 and 180 or a fragment thereof, thoseskilled in the art can routinely isolate a DNA with a high sequencesimilarity to these nucleotide sequences by using hybridizationtechniques and the like, and obtain proteins having the same NF-κ Bactivating activity as the protein having of an amino acid sequence ofany one of SEQ ID NO:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27,29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63,65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99,101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127,129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 152, 154, 156,158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178 and 180. Thus, thepresent invention also includes a protein that activates NF-κ B andcomprises an amino acid sequence having a high identity to the aminoacid sequence of any one of the above SEQ ID NO:1, 3, 5, 7, 9, 11, 13,15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49,51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85,87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117,119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145,147, 149, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174,176, 178 and 180. “High identity” refers to an amino acid sequencehaving an identity of at least 90%, preferably 95%, and more preferablyat least 97% over the entire length of an amino acid sequence expressedby any one of the above SEQ ID NO:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21,23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57,59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93,95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123,125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 152,154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178 and 180.The proteins of the present invention may be natural proteins derivedfrom any human or animal cells or tissues, chemically synthesizedproteins, or proteins obtained by genetic recombination techniques. Theprotein may or may not be subjected to post-translational modificationssuch as sugar chain addition or phosphorylation.

[0150] The present invention also includes a polynucleotide encoding theabove protein of the present invention. Examples of nucleotide sequencesencoding a protein consisting of an amino acid sequence of any one ofSEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31,33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67,69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101,103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129,131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 152, 154, 156, 158,160, 162, 164, 166, 168, 170, 172, 174, 176, 178 and 180 includenucleotide sequences of any one of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14,16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50,52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86,88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116,118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144,146, 148, 150, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171,173, 175, 177 and 179. The DNA includes cDNA, genomic DNA, andchemically synthesized DNA. In accordance with the degeneracy of thegenetic code, at least one nucleotide in the nucleotide sequenceencoding a protein consisting of an amino acid sequence of any one ofSEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31,33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67,69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101,103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129,131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 152, 154, 156, 158,160, 162, 164, 166, 168, 170, 172, 174, 176, 178 and 180 can besubstituted with other nucleotides without altering the amino acidsequence of the protein produced from the gene. Therefore, the DNAsequences of the present invention also include nucleotide sequencesaltered by substitution based on the degeneracy of the genetic code.Such DNA sequences can be synthesized using known methods.

[0151] The DNA of the present invention includes a DNA which encodes aprotein capable of activating NF-κ B and hybridizes under stringentconditions with the DNA sequence of the above nucleotide sequence of anyone of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28,30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64,66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100,102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128,130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 151, 153, 155,157, 159, 161, 163, 165, 167, 169, 171, 173, 175, 177 and 179. Stringentconditions are apparent to those skilled in the art, and can be easilyattained in accordance with various laboratory manuals such as T.Maniatis et al., Molecular Cloning A Laboratory Manual, Cold SpringHarbor Laboratory 1982, 1989.

[0152] That is, “stringent conditions” refer to overnight incubation at37° C. in a hybridization solution containing 30% formamide, 5×SSC (0.75M NaCl, 75 mM trisodium citrate), 5×Denhardt's solution, 0.5% SDS, 100μg/ml denatured, sheared salmon sperm DNA) followed by washing (threetimes) in 2×SSC, 0.1% SDS for 10 minutes at room temperature, thenfollowed by washing (two times) in 0.2×SSC, 0.1% SDS for 10 minutes at37° C. (low stringency). Preferred stringent conditions are overnightincubation at 42° C. in a hybridization solution containing 40%formamide, followed by washing (three times) in 2×SSC, 0.1% SDS for 10minutes at room temperature, then followed by washing (two times) in0.2×SSC, 0.1% SDS for 10 minutes at 42° C.(moderate stringency). Morepreferred stringent conditions are overnight incubation at 42° C. in ahybridization solution containing 50% formamide, followed by washing(three times) in 2×SSC, 0.1% SDS for 10 minutes at room temperature,followed by washing (two times) in 0.2×SSC, 0.1% SDS for 10 minutes at50° C. (high stringency). The DNA sequence thus obtained must encode aprotein capable of activating NF-κ B.

[0153] The present invention also includes a polynucleotide comprising anucleotide sequence which encodes a protein capable of activating NF-κ Band has a high sequence similarity to the nucleotide sequence of thepolynucleotide according to above item (3) or (4). Typically thesenucleotide sequence are 95% identical, preferably 97% identical, morepreferably 98-99% identical, most preferably at least 99% identical tothe nucleotide sequence of the polynucleotide according to above item(3) or (4) over the entire length thereof.

[0154] The above nucleotide sequence of the present invention can beused to produce the above protein using recombinant DNA techniques. Ingeneral, the DNA and peptide of the present invention can be obtainedby:

[0155] (A) cloning the DNA encoding the protein of the presentinvention;

[0156] (B) inserting the DNA encoding the entire coding region of theprotein or a part thereof into an expression vector to construct arecombinant vector;

[0157] (C) transforming host cells with the recombinant vector thusconstructed; and

[0158] (D) culturing the obtained cells to express the protein or itsanalogue, and then purifying it by column chromatography.

[0159] General procedures necessary to handle DNA and recombinant hostcells (e.g., E. coli) in the above steps are well known to those skilledin the art, and can be easily carried out in accordance with variouslaboratory manuals such as T. Maniatis et al., supra. All the enzymes,reagents, etc., used in these procedures are commercially available, andunless otherwise stated, such commercially available products can beused according to the use conditions specified by the manufacturer'sinstructions to attain completely its objects. The above steps (A) to(D) can be further illustrated in more details as follows.

[0160] Techniques for cloning the DNA encoding the protein of thepresent invention include, in addition to the methods described in thespecification of the present application, PCR amplification using asynthetic DNA having a part of the nucleotide sequence of the presentinvention (e.g., any one of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18,20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54,56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90,92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120,122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148,150, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173, 175,177 and 179) as a primer, and selection of the DNA inserted into asuitable vector by hybridization with a labeled DNA fragment encoding apartial or full coding region of the protein of the present invention ora labeled synthetic DNA. Another technique involves direct amplificationfrom total RNAs or mRNA fractions prepared from cells or tissues, usingthe reverse transcriptase polymerase chain reaction (RT-PCR method). Asa DNA inserted into a suitable vector, for example, a commerciallyavailable library (e.g., from CLONTECH and STRATAGENE) can be used.Techniques for hybridization are normally used in the art, and can beeasily carried out in accordance with various laboratory manuals such asT. Maniatis et al., supra. Depending on the intended purpose, the clonedDNA encoding the protein of the present invention can be used as such orif desired after digestion with a restriction enzyme or addition of alinker. The DNA thus obtained may have a nucleotide sequence of any oneof SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30,32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66,68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100,102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128,130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 151, 153, 155,157, 159, 161, 163, 165, 167, 169, 171, 173, 175, 177 and 179, or apolynucleotide of above items (3) to (7). The DNA sequence to beinserted into an expression vector in the above step (B) may be afull-length cDNA or a DNA fragment encoding the above full-lengthprotein, or a DNA fragment constructed so that it expresses a partthereof.

[0161] Thus, the present invention also includes a recombinant vector,which comprises the above DNA sequence. The expression vector for theprotein of the present invention can be produced, for example, byexcising the desired DNA fragment from the DNA encoding the protein ofthe present invention, and ligating the DNA fragment downstream of apromoter in a suitable expression vector.

[0162] Expression vectors for use in the present invention may be anyvectors derived from prokaryotes (e.g., E. coli), yeast, fungi, insectviruses and vertebrate viruses so long as such vectors are replicable.However, the vectors should be selected to be compatible withmicroorganisms or cells used as hosts. Suitable combinations of hostcell—expression vector systems are selected depending on the desiredexpression product.

[0163] When microorganisms are used as hosts, plasmid vectors compatiblewith these microorganisms are generally used as replicable expressionvectors for recombinant DNA molecules.

[0164] For example, the plasmids pBR322 and pBR327 can be used totransform E. coli. Plasmid vectors normally contain an origin ofreplication, a promoter, and a marker gene conferring upon a recombinantDNA a phenotype useful for selecting the cells transformed with therecombinant DNA. Example of such promoters include a β-lactamasepromoter, lactose promoter and tryptophan promoter. Examples of suchmarker genes include an ampicillin resistance gene, and a tetracyclineresistance gene. Examples of suitable expression vectors include theplasmids pUC18 and pUC19 in addition to pBR322, pBR327.

[0165] In order to express the DNA of the present invention in yeast,for example, YEp24 can be used as a replicable vector. The plasmid YEp24contains the URA3 gene, which can be employed as a marker gene. Examplesof promoters in expression vectors for yeast cells include promotersderived from genes for 3-phosphoglycerate kinase,glyceraldehyde-3-phosphate dehydrogenase and alcohol dehydrogenase.

[0166] Examples of promoters and terminators for use in expressionvectors to express the DNA of the present invention in fungal cellsinclude promoters and terminators derived from genes forphosphoglycerate kinase (PGK), glyceraldehyde-3-phosphate dehydrogenase(GAPD) and actin. Examples of suitable expression vectors include theplasmids pPGACY2 and pBSFAHY83.

[0167] Examples of promoters for use in expression vectors to expressthe DNA of the present invention in insect cells include a polyhedrinpromoter and P10 promoter.

[0168] Recombinant vectors used to express the DNA of the presentinvention in animal cells normally contain functional sequences toregulate genes, such as an origin of replication, a promoter to beplaced upstream of the DNA of the present invention, a ribosome-bindingsite, a polyadenylation site and a transcription termination sequence.Such functional sequences, which can be used to express the DNA of thepresent invention in eukaryotic cells, can be obtained from viruses andviral substances. Examples of such functional sequences include an SR αpromoter, SV40 promoter, LTR promoter, CMV (cytomegalovirus) promoterand HSV-TK promoter. Among them, a CMV promoter and SR α promoter can bepreferably used. As promoters to be placed inherently upstream of thegene encoding the protein of the present invention, any promoters can beused so long as they are suitable for use in the above host-vectorsystems. Examples of origins of replication include foreign origins ofreplication, for example, those derived from viruses such as adenovirus,polyoma virus and SV40 virus. When vectors capable of integration intohost chromosomes are used as expression vectors, origins of replicationof the host chromosomes may be employed. Examples of suitable expressionvectors include the plasmids pSV-dhfr (ATCC 37146), pBPV-1(9-1) (ATCC37111), pcDNA3.1 (INVITROGEN) and pME18S-FL3.

[0169] The present invention also includes a transformed cell, whichcomprises the above recombinant vector.

[0170] Microorganisms or cells transformed with the replicablerecombinant vector of the present invention can be selected fromremaining untransformed parent cells based on at least one phenotypeconferred by the recombinant vector. Phenotypes can be conferred byinserting at least one marker gene into the recombinant vector. Markergenes naturally contained in replicable vectors can be employed.Examples of marker genes include drug resistance genes such as neomycinresistance genes, and genes encoding dihydrofolate reductase.

[0171] As hosts for use in the above step (C), any of prokaryotes (e.g.,E. coli), microorganisms (e.g., yeast and fungi) as well as insect andanimal cells can be used so long as such hosts are compatible with theexpression vectors used. Examples of such microorganisms includeEscherichia coli strains such as E. coli K12 strain 294 (ATCC 31446), E.coli X1776 (ATCC 31537), E. coli C600, E. coli JM109 and E. coli Bstrain; bacterial strains belonging to the genus Bacillus such asBacillus subtilis; intestinal bacteria other than E. coli, such asSalmonella typhimurium or Serratia marcescens; and various strainsbelonging to the genus Pseudomonas. Examples of such yeast includeSaccharomyces cerevisiae, Schizosaccharomyces pombe, and Pichiapastoris. Examples of such fungi include Aspergillus nidulans, andAcremonium chrysogenum (ATCC 11550).

[0172] As insect cells, for example, Spodoptera frugiperda (Sf cells),High Five™ cells derived from eggs of Trichoplusiani, etc., can be usedwhen the virus is AcNPV. Examples of such animal cells include HEK 293cells, COS-1 cells, COS-7 cells, Hela cells, and Chinese hamster ovary(CHO) cells. Among them, CHO cells and HEK 293 cells are preferred.

[0173] When cells are used as hosts, combinations of expression vectorsand host cells to be used vary with experimental objects. According tosuch combinations, two types of expression (i.e. transient expressionand constitutive expression) can be included. “Transformation” ofmicroorganisms and cells in the above step (C) refers to introducing DNAinto microorganisms or cells by forcible methods or phagocytosis ofcells and then transiently or constitutively expressing the trait of theDNA in a plasmid or an intra-chromosome integrated form. Those skilledin the art can carry out transformation by known methods [see e.g.,“Idenshi Kougaku Handbook (Genetic Engineering Handbook)”, an extraissue of “Jikken Igaku (Experimental Medicine)”, YODOSHA CO., LTD.]. Forexample, in the case of animal cells, DNA can be introduced into cellsby known methods such as DEAE-dextran method, calcium-phosphate-mediatedtransfection, electroporation, lipofection, etc. For stable expressionof the protein of the present invention using animal cells, there is amethod in which selection can be carried out by clonal selection of theanimal cells containing the chromosomes into which the introducedexpression vectors have been integrated. For example, transformants canbe selected using the above selectable marker as an indication ofsuccessful transformation. In addition, the animal cells thus obtainedusing the selectable marker can be subjected to repeated clonalselection to obtain stable animal cell strains highly capable ofexpressing the protein of the present invention. When a dihydrofolatereductase (DHFR) gene is used as a selectable marker, one can cultureanimal cells while gradually increasing the concentration ofmethotrexate (MTX) and select the resistant strains, thereby amplifyingthe DNA encoding the protein of the present invention together with theDHFR gene to obtain animal cell strains having higher levels ofexpression.

[0174] The above transformed cells can be cultured under conditionswhich permit the expression of the DNA encoding the protein of thepresent invention to produce and accumulate the protein of the presentinvention. In this manner, the protein of the present invention can beproduced. Thus, the present invention also includes a process forproducing a protein, which comprises culturing a transformed cellcomprising the isolated polynucleotide according to above item (3) to(7) under conditions providing expression of the encoded protein andrecovering the protein from the culture.

[0175] The above transformed cells can be cultured by methods known tothose skilled in the art (see e.g., “Bio Manual Series 4”, YODOSHA CO.,LTD.). For example, animal cells can be cultured by various known animalcell culture methods including attachment culture such as Petri dishculture, multitray type culture and module culture, attachment culturein which cells are attached to cell culture carriers (microcarriers),suspension culture in which productive cells themselves are suspended.Examples of media for use in the culture include media commonly used foranimal cell culture, such as D-MEM and RPMI 1640.

[0176] In order to separate and purify the protein of the presentinvention from the above culture, suitable combinations of per se knownseparation and purification methods can be used. Examples such methodsinclude methods based on solubility, such as salting-out and solventprecipitation; methods based on the difference in charges, such asion-exchange chromatography; methods mainly based on the difference inmolecular weights, such as dialysis, ultrafiltration, gel filtration andSDS-polyacrylamide gel electrophoresis; methods based on specificaffinity, such as affinity chromatography; methods based on thedifference in hydrophobicity, such as reverse phase high performanceliquid chromatography; and methods based on the difference inisoelectric points, such as isoelectric focusing. For example, a proteinof the present invention can be recovered and purified from recombinantcell cultures by well-known methods including ammonium sulfate orethanol precipitation, acid extraction, anion or cation exchangechromatography, phosphocellulose chromatography, hydrophobic interactionchromatography, affinity chromatography, hydroxyapatite chromatographyand lectin chromatography. Most preferably, high performance liquidchromatography is employed for purification. Well known techniques forrefolding proteins may be employed to regenerate active conformationwhen the polypeptide is denatured during intracellular synthesis,isolation or purification.

[0177] The protein of the present invention can also be produced as afusion protein with another protein. These fusion proteins are alsoincluded within the present invention. For the expression of such fusionproteins, any vectors can be used so long as the DNA encoding theprotein can be inserted into the vectors and the vectors can express thefusion protein. Examples of proteins to which a polypeptide of thepresent invention can be fused include glutathione S-transferase (GST)and a hexa-histidine sequence (6×His). The fusion protein of the proteinof the present invention with another protein can be advantageouslypurified by affinity chromatography using a substance with an affinityfor the fusion partner protein. For example, fusion proteins with GSTcan be purified by affinity chromatography using glutathione as aligand.

[0178] Where the protein of the present invention is a membrane protein,a cell transformed with DNA encoding the above protein of the presentinvention can express the protein on/in a membrane thereof. A membranehaving the protein of the present invention being a membrane preparedfrom such a transformed cell, is within the scope of the presentinvention. It should be noted that herein, the term “membrane”encompasses a cell membrane, and all membranes of organelles. Thepreparation of membrane can be carried out by a method known in the art.For example, a membrane fraction can be obtained by culturingtransformant cells, recovering cells from the culture product,suspending the cells in a suitable buffer solution, disrupting the cellsusing an homogenizer or by adding glass beads and disrupting in avortex, removing undisrupted cells and the like by centrifugation,subjecting the supernatant to ultracentrifugation under suitableconditions, and suspending the obtained precipitate in a buffersolution. Conditions for ultracentrifugation can be determined asappropriate depending on the type of membrane etc.

[0179] The present invention also includes an inhibitory protein, i.e.,a protein capable of inhibiting the activity of the protein of aboveitem (1), (2) or (8). Examples of such inhibitory proteins includeantibodies, or other proteins that bind to active sites of a protein ofthe above item (1), (2) or (8), thereby inhibiting the expression oftheir activity.

[0180] The present invention also relates to an antibody thatspecifically binds the protein of the present invention or a fragmentthereof, and to production of such an antibody. The antibody is notspecifically limited so long as it can recognize the protein of thepresent invention. Examples of such antibodies include polyclonalantibodies, monoclonal antibodies and their fragments, single chainantibodies and humanized antibodies. Antibody fragments can be producedby known techniques. Examples of such antibody fragments include, butnot limited to, F(ab′)₂ fragments, Fab′ fragments, Fab fragments and Fvfragments. The antibody that specifically binds the protein of thepresent invention can be produced using the protein of the presentinvention or a peptide thereof as an immunogen according to per se knownprocess for producing antibodies or antisera. For example, a monoclonalor polyclonal antibody can be produced by administering the proteinaccording to above item (1) or (2) as an antigen or epitope-bearingfragments to a non-human animal. Such methods are described, forexample, in “Shin Idenshi Kougaku Handbook (New Genetic EngineeringHandbook)”, the third edition, an extra issue of “Jikken Igaku(Experimental Medicine)”, YODOSHA CO., LTD.

[0181] In the case of polyclonal antibodies, for example, the protein ofthe present invention or a peptide thereof can be injected to animalssuch as rabbits to produce antibodies directed against the protein orpeptide, and then their blood can be collected. The polyclonalantibodies can be purified from the blood, for example, by ammoniumsulfate precipitation or ion-exchange chromatography, or by using theaffinity column on which the protein has been immobilized.

[0182] In the case of monoclonal antibodies, for example, animals suchas mice are immunized with the protein of the present invention, theirspleen is removed and homogenized to obtain spleen cells, which are thenfused with mouse myeloma cells by using a reagent such as polyethyleneglycol. From the resulting hybrid cells (i.e. hybridoma cells), theclone producing the antibody directed against the protein of the presentinvention can be selected. Then, the resulting clonal hybridoma cellscan be implanted intraperitoneally into mice, the ascitic fluidrecovered from the mice. The resulting monoclonal antibody can bepurified, for example, by ammonium sulfate precipitation or ion-exchangechromatography, or by using the affinity column on which the protein hasbeen immobilized.

[0183] When the resulting antibody is used to administer it to humans,it is preferably used as a humanized antibody or human antibody in orderto reduce its immunogenicity. The humanized antibody can be producedusing transgenic mice or other mammals. For a general review of thesehumanized antibodies and human antibodies, see, for example, Morrison,S. L. et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984); Jones, P.T. et al., Nature 321:522-525 (1986); Hiroshi Noguchi, Igaku no Ayumi(J. Clin. Exp. Med.) 167:457-462 (1993); Takashi Matsumoto, Kagaku toSeibutsu (Chemistry and Biology) 36:448-456 (1998). Humanized chimericantibodies can be produced by linking a V region of a mouse antibody toa C region of a human antibody. Humanized antibodies can be produced bysubstituting a sequence derived from a human antibody for a region otherthan a complementarity-determining region from a mouse monoclonalantibody. In addition, human antibodies can be directly produced in thesame manner as the production of conventional monoclonal antibodies byimmunizing the mice whose immune systems have been replaced with humanimmune systems. These antibodies can be used to isolate or to identifyclones expressing the protein or to purify the protein of the presentinvention from a cell extract or transformed cells producing the proteinof the present invention. These proteins can also be used to constructELISA, RIA (radioimmunoassay) and western blotting systems. These assaysystems can be used for diagnostic purposes for detecting an amount ofthe protein of the present invention present in a body sample in atissue or a fluid in the blood of an animal, preferably human. Forexample, they can be used for diagnosis of a disease characterized byundesirable activation of NF-κ B resulting from (expression) abnormalityof the protein of the present invention, such as inflammation,autoimmune disease, infection (for example, HIV infection), bonedisease, cancer and the like. In order to provide a basis for diagnosisof a disease, a standard value must be established. However, this is awell-known technique to those skilled in the art. For example, a methodof calculating the standard value comprises binding a body fluid or acell extract of normal individual of a human or an animal to an antibodyagainst the protein of the present invention under a suitable conditionfor the complex formation, detecting the amount of the antibody-proteincomplex by chemical or physical means and then calculating the standardvalue for the normal sample using a standard curve prepared from astandard solution containing a known amount of an antigen (the proteinof the present invention). The presence of a disease can be confirmed bydeviation from the standard value obtained by comparison of the standardvalue with the value obtained from a sample of an individual latentlysuffering from a disease associated with the protein of the presentinvention. These antibodies can also be used as reagents for studyingfunctions of the protein of the present invention.

[0184] The antibody of the present invention can be used as a medicamentas follows. Where the antibody of the present invention is used as amedicament, it is preferable to use an antibody which can inhibit theaction of activating NF-κ B possessed by the protein of the presentinvention (i.e. neutralizing antibody).

[0185] The antibodies of the present invention can be purified and thenadministered to patients of a disease characterized by undesirableactivation of NF-κ B resulting from (expression) abnormality of theprotein of the present invention, such as inflammation, autoimmunedisease, infection (such as HIV infection), bone disease, cancer and thelike. Thus in another aspect, the present invention is a pharmaceuticalcomposition which comprises the above antibody as an active ingredient,and therapy using the antibody of the present invention. In suchpharmaceutical compositions, the active ingredient may be combined withother therapeutically active ingredients or inactive ingredients (e.g.,conventional pharmaceutically acceptable carriers or diluents such asimmunogenic adjuvants) and physiologically non-toxic stabilizers andexcipients. The resulting combinations can be sterilized by filtration,and formulated into vials after lyophilization or into various dosageforms in stabilized and preservable aqueous preparations. Administrationto a patient can be intra-arterial administration, intravenousadministration and subcutaneous administration, which are well known tothose skilled in the art. The dosage range depends upon the weight andage of the patient, route of administration and the like. Suitabledosages can be determined by those skilled in the art. These antibodiesexhibit therapeutic activity by inhibiting the NF-κ B activationmediated by the protein of the present invention.

[0186] The DNA of the present invention can also be used to isolate,identify and clone other proteins involved in intracellular signaltransduction processes. For example, the DNA sequence encoding theprotein of the present invention can be used as a “bait” in yeasttwo-hybrid systems (see e.g., Nature 340:245-246 (1989)) to isolate andclone the sequence encoding a protein (“prey”) which can associate withthe protein of the present invention. In a similar manner, it can bedetermined whether the protein of the present invention can associatewith other cellular proteins (e.g., NIK and TRAF2). In another method,proteins which can associate with the protein of the present inventioncan be isolated from cell extracts by immunoprecipitation [see e.g.,“Shin Idenshi Kougaku Handbook (New Genetic Engineering Handbook)”, anextra issue of “Jikken Igaku (Experimental Medicine)”, YODOSHA CO.,LTD.] using antibodies directed against the protein of the presentinvention. In still another method, the protein of the present inventioncan be expressed as a fusion protein with another protein as describedabove, and immunoprecipitated with an antibody directed against thefusion protein in order to isolate a protein which can associate withthe protein of the present invention.

[0187] The diagnostic assays offer a process for diagnosing diseases ordetermining a susceptibility to the diseases through detection ofmutation in a gene for a protein according to item (1), (2) or (8) whichhas a function of activating NF-κ B, by the methods described. Inaddition, such diseases may be diagnosised by methods comprisingdetermining from a sample derived from a subject an abnormally decreasedor increased level of protein or mRNA. Decreased or increased expressioncan be measured at the RNA level using any of the methods well known inthe art for the quantitation of polynucleotides, for example, nucleicacid amplification methods such as RT-PCR, and methods such as RNaseprotection assay, Northern blotting and other hybridization methods.Assay techniques that can be used to determine levels of a protein in asample derived from a host are well-known to those skilled in the art.Such assay methods include radioimmunoassays, competitive-bindingassays, Western blot analysis and ELISA assays. The DNA of the presentinvention can be used to detect abnormality in the DNA or mRNA encodingthe protein of the present invention or a peptide fragment thereof. Theinvention relates to a method for diagnosing a disease, orsusceptibility to a disease associated with the expression of theprotein according to above item (1), (2) or (8) in a subject, whichcomprises determining mutations in the polynucleotide sequence encodingthe protein. Thus, for example, the DNA of the present invention isuseful for gene diagnosis regarding damage, mutations, and reduced,increased or over-expression of the DNA or mRNA. That is, the presentinvention includes a method for diagnosing a disease or susceptibilityto a disease associated with the expression or activity of NF-κ B in asubject, which comprises the steps of:

[0188] (a) determining the presence or absence of a mutation in thenucleotide sequence encoding the protein according to any one of aboveitem (1), (2) or (8), in the genome of the subject, and/or

[0189] (b) analyzing the amount of expression of said protein in asample derived from said subject, wherein a diagnosis of disease ispreferably made when the amount of the protein expressed is 2-fold orhigher than normal, or half or lower than normal.

[0190] When the nucleotide sequence encoding the protein of above item(1), (2) or (8) which has a function of activating NF-κ B, contains amutation according to the above step (a), the mutation may cause adisease associated with NF-κ B activation. When the amount of theexpression of the protein of above item (1), (2) or (8) is differentfrom the normal value according to the above step (b), the abnormalexpression of the novel protein of the present invention which acts toactivate NF-κ B may be responsible for diseases associated with NF-κ Bactivation. In the above step (a), determination of the presence orabsence of a mutation in the nucleotide seqeunce of a the gene encodingthe protein of above item (1), (2) or (8) which has a function ofactivating NF-κ B, may involve RT-PCR using a part of the nucleotidesequences of genes encoding these proteins as a primer, followed byconventional DNA sequencing to detect the presence or absence of themutation. PCR-SSCP [Genomics 5:874-879 (1989); “Shin Idenshi KougakuHandbook (New Genetic Engineering Handbook)”, an extra issue of “JikkenIgaku (Experimental Medicine)”, YODOSHA CO., LTD.] can also be used todetermine the presence or absence of the mutation.

[0191] Measurement of the amount of the expression of the protein in theabove step (b) may involve, for example, using the antibody of aboveitem (18) or (19).

[0192] The present invention also relates to a method for screeningcompounds which inhibit or promote NF-κ B activation using the proteinsof the invention, which comprises the steps of:

[0193] (a) providing a cell with a gene encoding a protein thatactivates NF-κ B, and a component that provides a detectable signal uponactivation of NF-κ B;

[0194] (b) culturing the transformed cell under conditions, which permitthe expression of the gene in the transformed cell;

[0195] (c) contacting the transformed cell with one or more compounds;and

[0196] (d) measuring the detectable signal; and

[0197] (e) isolating or identifying an activator compound and/or aninhibitor compound by measuring the detectable signal.

[0198] Further, it is preferable to isolate or identify as an activatorcompound, a compound that increases said detectable signal 2-fold orhigher than normal, and to isolate or identify as an inhibitor compound,a compound that decreases said detectable signal half or less thannormal.

[0199] Examples of components capable of providing a detectable signalinclude reporter genes. Reporter genes are used instead of directlydetecting the activation of transcription factors of interest. Thetranscriptional activity of a promoter of a gene is analyzed by linkingthe promoter to a reporter gene and measuring the activity of theproduct of the reporter gene (“Bio Manual Series 4” (1994), YODOSHA CO.,LTD.).

[0200] Any peptide or protein can be used so long as those skilled inthe art can measure the activity or amount of the expression product(including the amount of the produced mRNA) of the reporter genes. Forexample, enzymatic activity of chloramphenicol acetyltransferase,β-galactosidase, luciferase, etc., can be measured. Any reporterplasmids can be used to evaluate NF-κ B activation so long as thereporter plasmids have an NF-κ B recognition sequence inserted upstreamof the reporter gene. For example, pNF-κ B-Luc (STRATAGEGE) can be used.Other examples include NF-κ B dependent reporter plasmids described inTanaka S. et al., J. Vet. Med. Sci. Vol.59 (7); Rothe M. et al., ScienceVol.269, p.1424-1427 (1995).

[0201] Any host cells may be used so long as NF-κ B activation can bedetected in the host cells. Preferred host cells are mammalian cellssuch as 293-EBNA cells. Transformation and culture of the cells can becarried out as described above.

[0202] In a specific embodiment, the method for screening a compoundwhich inhibits or promotes NF-κ B activation comprises culturing thetransformed cell for a certain period of time, adding a certain amountof a test compound, measuring the reporter activity expressed by thecell after a certain period of time, and comparing the activity withthat of a cell to which the test compound has not been added. Thereporter activity can be measured by methods known in the art (see e.g.,“Bio Manual Series 4” (1994), YODOSHA CO., LTD.). Examples of testcompounds include, but not limited to, low molecular weight compoundsand peptides. Test compounds may be artificially synthesized compoundsor naturally occurring compounds. Test compounds may be a singlecompound or mixtures. Examples of such detectable signals which may bemeasured include the amount of mRNA or proteins for genes whoseexpression is known to be induced by NF-κ B activation (e.g., genes forIL-1 and TNF-α) in addition to the above reporter genes. The amount ofmRNA can be measured, for example, by northern hybridization, RT-PCR,etc. The amount of proteins can be measured, for example, by usingantibodies. The antibodies may be produced by known methods.Commercially available antibodies(from, e.g., Wako Pure ChemicalIndustries, Ltd.) can also be used.

[0203] It is also possible to produce a pharmaceutical compositionaccording to the following steps (a) to (f):

[0204] (a) providing a cell with a gene encoding a protein thatactivates NF-κ B, and a component that provides a detectable signal uponactivation of NF-κ B;

[0205] (b) culturing the transformed cell under conditions, which permitthe expression of the gene in the transformed cell;

[0206] (c) contacting the transformed cell with one or more candidatecompounds;

[0207] (d) measuring the detectable signal;

[0208] (e) isolating or identifying an activator compound and/or aninhibitor compound by measuring the detectable signal; and

[0209] (f) optimizing the isolated or identified compound as apharmaceutical composition.

[0210] Further, it is preferable to isolate or identify as an activatorcompound, a compound that increases said detectable signal 2-fold orhigher than normal, and to isolate or identify as an inhibitor compound,a compound that decreases said detectable signal half or less thannormal.

[0211] The protein of the present invention may also be used in a methodfor the structure-based design of an agonist, antagonist or inhibitor ofthe protein, by:

[0212] (a) determining in the first instance the three-dimensionalstructure of the protein;

[0213] (b) deducing the three-dimensional structure for the likelyreactive or binding site(s) of an agonist, antagonist or inhibitor;

[0214] (c) synthesising candidate compounds that are predicted to bindto or react with the deduced binding or reactive site; and

[0215] (d) testing whether the candidate compounds are indeed agonists,antagonists or inhibitor.

[0216] The present invention also includes a compound obtainable by theabove screening method. However, the screening method of the presentinvention is not limited to the above method. The present invention alsoincludes a process for producing the pharmaceutical composition by themethod of above item (16).

[0217] There is no special limitation to the above candidate compounds.Such compounds include low molecular weight compounds and peptides. Theymay be artificially synthesised compounds and naturally occurringcompounds. As the compounds obtained by the above screening methods havea function of inhibiting or promoting NF-κ B activation, they are usefulas therapeutic or preventive pharmaceuticals for the treament ofdiseases resulting from unfavorable activation or inactivation of NF-κB. In order to isolate and purify the target compounds from the mixture,it is suitable to combine the known methods such as filtration,extraction, washings, drying, concentration, crystallization,variouschromatography. When obtainment of a salt of the compounds is desired, acompound which is obtained in the form of a salt can be purified as itis. A compound which is obtained in the free form can be converted intoa salt by isolating and purifying a salt obtained by dispersing ordissolving the compound into a suitable solvent and then adding adesired acid or base. Examples of a step to optimize the compounds orsalts thereof obtained by the method of the present invention as apharmaceutical composition, include methods of formulating according toordinary processes such as the following. The above compounds or theirpharmaceutically acceptable salts in an amount effective as an activeingredient, and pharmaceutically acceptable carriers can be mixed. Aform of formulation suitable for the mode of administration is selected.A composition suitable for oral administration includes a solid formsuch as tablet, granule, capsule, pill and powder, and solution formsuch as solution, syrup, elixir and dispersion. A form useful forparenteral administration includes sterile solution, dispersion,emulsion and suspension. The above carriers include, for example, sugarssuch as gelatin, lactose and glucose, starches such as corn, wheat, riceand maize, fatty acids such as stearic acid, salts of fatty acids suchas calcium stearate, magnesium stearate, talc, vegetable oil,alcoholsuch as stearyl alcohol and benzyl alcohol, gum, and polyalkyleneglycol. Examples of such liquid carriers include generally water,saline, sugar solution of dextrose and the lile, glycols such asethylene glycol, propylene glycol and polyethylene glycol.

[0218] The present invention also includes a kit for screening compoundsfor activity to inhibit or promote NF-κ B activation. The kit comprisesreagents and the like necessary for screening compounds for inhibitingor promoting activity for NF-κ B activation, including:

[0219] (a) a cell comprising a gene encoding a protein that activatesNF-κ B, and a component thst provides a detectable signal enablingdetection of NF-κ B activation after activation of NF-κ B; and

[0220] (b) reagents for measuring the detectable signal.

[0221] In another aspect, the present invention relates to a diagnostickit which comprises:

[0222] (a) a polynucleotide of the present invention having a nucleotidesequence expressed by SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22,24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58,60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94,96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124,126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 151,153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173, 175, 177 or 179;

[0223] (b) a nucleotide sequence complementary to that of (a);

[0224] (c) a protein of the present invention having an amino acidseqeunce expressed by SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21,23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57,59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93,95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123,125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 152,154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178 or 180,or a fragment thereof; or

[0225] (d) an antibody to the protein of the present invention of (c).

[0226] A kit comprising at least one of (a) to (d) is useful fordiagnosing a disease or susceptibility to a disease such asinflammation, autoimmune diseases, infectious diseases (e.g., HIVinfection) and cancers.

[0227] Because NF-κ B is involved in a wide variety of pathologicalconditions such as inflammation, autoimmune diseases, cancers and viralinfections, it is an attractive target for drug design and therapeuticintervention. Many experiments show that NF-κ B activity may havesignificant physiological effects [see e.g., Ann. Rheum. Ds. 57:738-741(1998); American Journal of Pathology 152:793-803 (1998); ARTHRITIS &RHEUMATISM 40:226-236 (1997); Am. J. Respir. Crit. Care Med.158:1585-1592 (1998); J. Exp. Med. 188:1739-1750 (1998); Gut 42:477-484(1998); The Journal of Immunology 161:4572-4582 (1998); Nature Medicine3:894-899 (1997)]. The finding of the new protein described hereincapable of activating NF-κ B has provided a new method for controllingan abnormal NF-κ B function. Thus, the present invention also relates touse of a compound which inhibits the function of the protein capable ofactivating NF-κ B described above, for inhibiting NF-κ B activation.Further, the present invention relates to a method of using a compoundwhich activates the function of the protein capable of activating NF-κ Bdescribed above, for promoting NF-κ B activation. The compound obtainedby the above screening method, which inhibits NF-κ B activation, isuseful as a medicament to treat or prevent diseases characterized byundesirable activation of NF-κ B, such as inflammation, autoimmunediseases (such as rheumatoid arthritis, systemic lupus erythematosus,asthma, etc), infectious diseases, bone diseases, and graft rejection.Recently, it has also become apparent that NF-κ B activation controlsapoptosis of cells. The compound obtained by the above screening method,which inhibits NF-κ B activation, may be capable of stimulatingapoptosis. Diseases which may be treated by the induction of apoptosisinclude tumors.

[0228] Further, examples of diseases related to abnormality in NF-κ Bactivation include AIDS (acquired immunodeficiency syndrome),neurodegenerative diseases (Alzheimer's disease, Parkinson's disease,amyotrophic lateral sclerosis, etc.) ischemic disorders (i.e. thosecaused by cardiac infarction, reperfusion injury, etc), myelogenesisincompetency syndrome (aplastic anemia, etc), skin diseases (Toxicepidermal necrolysis, etc), proliferative nephritis (IgA nephritis,purpuric nephritis, lupus nephritis, etc) and fulminant hepatitis. Thus,a compound obtained by the above screening method, which inhibits orpromotes NF-κ B activation, is useful as a medicament to treat orprevent these diseases.

[0229] In addition, the gene encoding the protein of the presentinvention is useful for gene therapy to treat various diseases such ascancers, autoimmune diseases, allergy diseases and inflammatoryresponse. “Gene therapy” refers to administering into the human body agene or a cell into which a gene has been introduced. The protein of thepresent invention and the DNA encoding the protein can also be used fordiagnostic purposes. That is, according to the present invention, thereis provided a gene therapy agent comprising a gene encoding the proteinof the present invention.

[0230] Further, where a gene encoding the protein of the presentinvention is used for gene therapy, the RNA interference (RNAi) method,which is described later, can be applied. That is, according to thepresent invention, there is also provided a vector for gene therapy thatcan express a double stranded nucleic acid having a nucleotide sequenceencoding the protein of the present invention. Further, the presentinvention encompasses a gene therapy agent comprising one or more doublestranded nucleic acids having a nucleotide sequence encoding the proteinof the present invention, and/or one or more vectors for gene therapyuse which express a double stranded nucleic acid having a nucleotidesequence encoding the protein of the present invention.

[0231] Forms of the agent for gene therapy, are not particularlylimited, and examples include a pharmaceutical composition wherein anexpression vector including the gene of the present invention iscontained in a pharmaceutical carrier consisting of physiological buffersolution. A pharmaceutical carrier can otherwise include a suitablestabilizing agent (e.g. nuclease inhibitor, etc.), chelating agent (e.g.EDTA, etc.) and/or other excipients. Further, the agent for gene therapycan be supplied as a complex of the double stranded nucleic acid of thepresent invention and/or the expression vector of the present inventionwith a liposome. The gene therapy agent can be administered, forexample, by using a catheter. The gene therapy agent of the presentinvention can also, for example, be directly injected into a patient'sblood vessel.

[0232] The dose of the gene therapy agent of the present invention to beadministered should be increased or decreased as appropriate dependingon conditions such as the age, sex, weight, and condition of thepatient, and administration route, etc, however, generally, the dose fora single administration to an adult is within a range of about 1 μg/kgto 1000 mg/kg, and preferably within a range of 10 μg/kg to 100 mg/kg,as an amount of DNA, being the effective component. There is nolimitation on number of administrations. The present invention alsoencompasses a method where one or more the double stranded nucleic acidsof the present invention and/or one or more the expression vectors ofthe present invention, are selected and administered simultaneously orsequentially.

[0233] The compound obtained by the screening method of the presentinvention or a salt thereof can be formulated into the abovepharmaceutical compositions (e.g., tablets, capsules, elixirs,microcapsules, sterile solutions and suspensions) according toconventional procedures. The formulations thus obtained are safe and oflow toxicity, and can be administered, for example, to humans andmammals (e.g., rats, rabbits, sheep, pigs, cattle, cats, dogs andmonkeys). Administration to patients can be carried out by methods knownin the art, such as intra-arterial injection, intravenous injection andsubcutaneous injection. The dosage may vary with the weight and age ofthe patient as well as a mode of administration, but those skilled inthe art can appropriately select suitable dosages. When the compound canbe encoded by DNA, the DNA can be inserted into a vector for genetherapy, and gene therapy can be carried out. The dosage and mode ofadministration may vary with the weight, age and symptoms of thepatient, but those skilled in the art can appropriately select them.Thus, the present invention also relates to a pharmaceutical compositionwhich comprises the above compound as an active ingredient.

[0234] In addition, the above compound is useful as a medicament totreat or prevent diseases characterized by abnormal NF-κ B activity,such as inflammation, autoimmune diseases, viral diseases, infectiousdiseases, cancers and bone diseases. Thus, the present invention alsorelates to a pharmaceutical composition for inflammation, autoimmunediseases, viral diseases, infectious diseases, cancers, bone diseases,etc., which comprises the above compound. Specifically, the compound isuseful as a therapeutic and/or prophylactic drug against, for example,rheumatoid arthritis, osteoarthritis, systemic lupus erythematosus,diabetes, sepsis, asthma, allergic rhinitis, ischemic heart diseases,inflammatory intestinal diseases, subarachnoid hemorrhage, viralhepatitis, AIDS, atherosclerosis, atopic dermatosis, viral infections,Crohn's disease, diabetes, gout, hepatitis, multiple sclerosis, cardiacinfarction, nephritis, osteoporosis, Alzheimer's, Parkinson's disease,Huntington's chorea, psoriasis, amyotrophic lateral sclerosis, oraplastic anemia.

[0235] The present invention also relates to the use of a pharmaceuticalcomposition produced according to above item (16) for manufacturing amedicament against inflammation, autoimmune diseases, viral diseases,cancers, infectious diseases, bone diseases, etc. The present inventionalso includes an antisense oligonucleotide against a gene of any one ofabove items (3) to (7). An antisense oligonucleotide refers to anoligonucleotide complementary to the target gene sequence. The antisenseoligonucleotide can inhibit the expression of the target gene byinhibiting RNA functions such as translation to proteins, transport tothe cytoplasm and other activity necessary for overall biologicalfunctions. In this case, the antisense oligonucleotide may be RNA orDNA. The DNA sequence of the present invention can be used to produce anantisense oligonucleotide capable of hybridizing with the mRNAtranscribed from the gene encoding the protein of the present invention.It is known that an antisense oligonucleotide. generally has aninhibitory effect on the expression of the corresponding gene (see e.g.,Saibou Kougaku Vol.13, No.4 (1994)). The oligonucleotide containing anantisense coding sequence against a gene encoding the protein of thepresent invention can be introduced into a cell by standard methods. Theoligonucleotide effectively blocks the translation of mRNA of the geneencoding the protein of the present invention, thereby blocking itsexpression and inhibiting undesirable activity.

[0236] The antisense oligonucleotide of the present invention may be anaturally occurring oligonucleotide or its modified form [see e.g.,Murakami & Makino, Saibou Kougaku Vol.13, No.4, p.259-266 (1994); AkiraMurakami, Tanpakushitsu Kakusan Kouso (PROTEIN, NUCLEIC ACID AND ENZYME)Vol.40, No.10, p.1364-1370 (1995),Tunenari Takeuchi et al., Jikken Igaku(Experimental Medicien) Vol. 14, No. 4 p85-95(1996)]. Thus, theoligonucleotide may have modified sugar moieties or inter-sugarmoieties. Examples of such modified forms include phosphothioates andother sulfur-containing species used in the art. According to severalpreferred embodiments of the present invention, at least onephosphodiester bond in the oligonucleotide is substituted with thestructure which can enhance the ability of the composition to permeatecellular regions where RNA with the activity to be regulated is located.

[0237] Such substitution preferably involves a phosphorothioate bond, aphosphoramidate bond, methylphosphonate bond, or a short-chain alkyl orcycloalkyl structure. The antisense oligonucleotide may also contain atleast some modified base forms. Thus, it may contain purine andpyrimidine derivatives other than naturally occurring purine andpyrimidine. Similarly, the furanosyl moieties of the nucleotide subunitscan be modified so long as the essential purpose of the presentinvention is attained. Examples of such modifications include 2′-O-alkyland 2′-halogen substituted nucleotides. Examples of modifications insugar moieties at their 2-position include OH, SH, SCH₃, OCH₃, OCN orO(CH₂)_(n)CH₃, wherein n is 1 to about 10, and other substituents havingsimilar properties. All the analogues are included in the scope of thepresent invention so long as they can hybridize with the mRNA of thegene of the present invention to inhibit functions of the mRNA.

[0238] The antisense oligonucleotide of the present invention containsabout 3 to about 50 nucleotides, preferably about 8 to about 30nucleotides, more preferably about 12 to about 25 nucleotides. Theantisense oligonucleotide of the present invention can be produced bythe well-known solid phase synthesis technique. Devices for suchsynthesis are commercially available from some manufactures includingApplied Biosystems. Other oligonucleotides such as phosphothioates canalso be produced by methods known in the art.

[0239] The antisense oligonucleotide of the present invention isdesigned to hybridize with the mRNA transcribed from the gene of thepresent invention. Those skilled in the art can easily design anantisense oligonucleotides based on a given gene sequence (For example,Murakami and Makino: Saibou Kougaku Vol. 13 No.4 p259-266 (1994), AkiraMurakami: Tanpakushitsu Kakusan Kouso (PROTEIN, NUCLEIC ACID AND ENZYME)Vol. 40 No.10 p1364-1370 (1995), Tunenari Takeuchi et al., Jikken Igaku(Experimental Medicine) Vol. 14 No. 4 p85-95 (1996)). Recent studysuggests that antisense oligonucleotides which are designed in a regioncontaining 5′ region of mRNA, preferably, the translation initaiationsite, are most effective for the inhibition of the expression of a gene.The length of the antisense oligonucleotides is preferably 15 to 30nucleotides and more preferably 20 to 25 nucleotides. It is important toconfirm no interaction with other mRNA and no formation of secondarystructure in the oligonucleotide sequence by homology search. Theevaluation of whether the designed antisense oligonucleotide isfunctional or not can be determined by introducing the antisenceoligonucleotide into a suitable cell and measuring the amount of thetarget mRNA, for example by northern blotting or RT-PCR, or the amountof the target protein, for example by western blotting or fluorescentantibody technique, to confirm the effect of expression inhibition.

[0240] Another method includes the triple helix technique. Thistechnique involves forming a triple helix on the targeted intra-nuclearDNA sequence, thereby regulating its gene expression, mainly at thetranscription stage. The antisense oligonucleotide is designed mainly inthe gene region involved in the transcription and inhibits thetranscription and the production of the protein of the presentinvention. Such RNA, DNA and oligonucleotide can be produced using knownsynthesizers.

[0241] The antisense oligonucleotide may be introduced into the cellscontaining the target nucleic acid sequence by any of DNA transfectionmethods such as calcium phosphate method, electroporation,lipofection,microinjection, or gene transfer methods including the use of genetransfer vectors such as viruses. An antisense oligonucleotideexpression vector can be prepared using a suitable retrovirus vector,then the expression vector can be introduced into the cells containingthe target nucleic acid sequence by contacting the vector with the cellsin vivo or ex vivo.

[0242] The DNA of the present invention can be used in the antisenseRNA/DNA technique or the triple helix technique to inhibit NF-κ Bactivation mediated by the protein of the present invention.

[0243] Further, polypeptides encoded by the polynucleotides of SEQ IDNOS: 6, 88, 153 and 161 are explained below in detail, as examples.However, these do not limit the present invention in any way.

[0244] As a result of preparing an expression vector for a fusionprotein with GFP and expressing in Vero cells or COS 7 cells, thepolypeptide encoded by the polynucleotide of SEQ ID NO: 6, was found tobe primarily localized in endoplasmic reticulum. Further, as a result ofexamination of expression in human tissues (bone marrow, brain, colon,heart, kidney, leukocyte (granule), leukocyte (resting lymph node),thymus, spleen, small intestine, trachea, liver, lung, skin, adrenalgland, salivary gland, testis and uterus) by Real-time PCR, highexpression of the polypeptide was primarily observed in leukocyte(granule), spleen, lung and uterus.

[0245] Therefore, the polynucleotide of SEQ ID NO: 6 and the polypeptideencoded by the polynucleotide are useful as reagents for identificationof tissues or cell types present in a biological sample, and fordiagnosis of a disease or condition including (but not limited to)disorders of leukocyte, spleen, lung and uterus.

[0246] Further, the tissue distribution thereof indicates that thepolynucleotide and polypeptide of the present invention are useful inthe diagnosis and treatment of various immune system disorders (forexample, infection, inflammation, allergy, immune deficiency and thelike), and lung-related disorders (for example, lung cancer, infection,asthma, and the like).

[0247] The polypeptide encoded by the polynucleotide of SEQ ID NO: 88possesses a sequence having homology to TIR (Toll/IL-1 receptor) domainsequence, and interacts with at least Toll-like receptor (TLR) 3, TLR4and TLR9. Further, as a result of preparing an expression vector for afusion protein with GFP, and expressing in Vero cells or COS 7 cells,the polypeptide was primarily localized in cell membrane. Further, as aresult of examination of expression in human tissues (bone marrow,brain, colon, heart, kidney, leukocyte (granule), leukocyte (restinglymph node), thymus, spleen, small intestine, trachea, liver, lung,skin, adrenal gland, salivary gland, testis and uterus) using Real-timePCR, high expression of the polypeptide was primarily observed inleukocyte (granule), spleen, lung and uterus.

[0248] Therefore, the polynucleotide of SEQ ID NO: 88 and thepolypeptide encoded by the polynucleotide are useful as reagents foridentification of tissues or cell types present in a biological sample,and for diagnosis of a disease or condition including (but not limitedto) disorders of leukocyte, spleen, lung and uterus.

[0249] Further, the tissue distribution thereof indicates that thepolynucleotide of SEQ ID NO: 88 and the polypeptide are useful in thediagnosis and treatment of various immune system disorders (for example,infection, inflammation, allergy, immune deficiency and the like), andlung-related disorders (for example, lung cancer, infection, asthma, andthe like).

[0250] Further, the fact that the polypeptide of the present inventioninteracts with TLR, together with intracellular localization results,indicate that the polypeptide is deeply involved in immune system,particularly signal transduction in innate immune system, and that thepolynucleotide and polypeptide of the present invention are useful fordiagnosis and treatment of various immune system disorders. Inparticular, they are useful in the diagnosis and treatment of immunesystem disorders related to chronic or acute microorganism infections(e.g. bacterial, fungal or viral infection).

[0251] As a result of examination of expression in human tissues (bonemarrow, brain, colon, heart, kidney, leukocyte (granule), leukocyte(resting lymph node), thymus, spleen, small intestine, trachea, liver,lung, skin, adrenal gland, salivary gland, testis and uterus) usingReal-time PCR, high expression of the polypeptide encoded by thepolynucleotide of SEQ ID NO: 153 was primarily observed in lung andspleen. Further, the polypeptide encoded by the polynucleotide of SEQ IDNO: 153 possesses a sequence having homology to TIR (Toll/IL-1 receptor)domain sequence, and as a result of cotransfecting EBNA cells with avector for expression of the polypeptide of SEQ ID NO: 153 and areporter plasmid for human interferon (IFN)-β gene promoter, inductionof expression of IFNβ promoter reporter was observed. The induction ofIFNβ expression can be measured by a method using a reporter plasmid ora method comprising the measurement of the amount of mRNA or protein ofIFNβ. The amount of mRNA can be measured by, for example, Northernhybridization or RT-PCR. The amount of protein can be measured by, forexample, a method using an antibody or ELISA. Antibodies can be preparedby a method known in the art, or obtained from manufacturer (forexample, FUNAKOSHI or BioSource International).

[0252] Therefore, the polynucleotide of SEQ ID NO: 153 and thepolypeptide encoded by the polynucleotide are useful as reagents foridentification of tissues or cell types present in a biological sample,and for diagnosis of a disease or condition including (but not limitedto) disorders of lung and spleen.

[0253] Further, the tissue distribution thereof indicates that thepolynucleotide and polypeptide of the present invention are useful inthe diagnosis and treatment of various immune system disorders (forexample, infection, inflammation, allergy, immune deficiency and thelike), and lung-related disorders (for example, lung cancer, infection,asthma, and the like).

[0254] Further, the fact that the polypeptide of the present inventionpossesses a TIR domain sequence and is a polypeptide involved inexpression induction of IFN-β indicates that the polypeptide of thepresent invention is involved in signal transduction in the innateimmune system, and that the polynucleotide and polypeptide of thepresent invention are useful for diagnosis and treatment of variousimmune system disorders. In particular, it is possible that they areuseful for the diagnosis and treatment of disorders of the immune systemrelated to chronic or acute microorganism infections (for example,bacterial, fungal or viral infection).

[0255] It should be noted that the reporter plasmid for human IFN-β genepromoter was prepared in the following method. Primers of two syntheticoligonucleotides: 5′-CTAGCTAGCTAGAAACTACTAAAATGTAAATGACATAG-3′ (SEQ IDNO: 183) and 5′-CGCAAGCTTGAAAGGTTGCAGTTAGAATGTCCTTTC-3′ (SEQ ID NO:184), were designed, and using this primer pair, PCR was performed usinghuman genome (CLONTECH) as a template. An amplified fragment of approx.0.15 kb was isolated, and after digesting with NheI and HindIIIrestriction enzymes, and the fragment was inserted between the NheI siteand HindIII site of firefly luciferase reporter vector pGL3-Basic Vector(Promega Corporation) using T4 DNA ligase to prepare the plasmid.

[0256] As a result of examining expression of the gene of SEQ ID NO: 161in human tissues (bone marrow, brain, colon, heart, kidney, leukocyte(granule), leukocyte (resting lymph node), thymus, spleen, smallintestine, trachea, liver, lung, skin, adrenal gland, salivary gland,testis, uterus) by RT-PCR, high expression was observed primarily inlung.

[0257] Therefore, the polynucleotide of SEQ ID NO: 161 and thepolypeptide encoded by the polynucleotide are useful as reagents foridentification of tissues or cell types present in a biological sample,and for diagnosis of a disease or condition including (but not limitedto) disorders of the lung.

[0258] Further, the tissue distribution thereof indicates that thepolynucleotide and polypeptide of the present invention are useful inthe diagnosis and treatment of lung-related disorders (for example, lungcancer, infection due to bacteria and virus, asthma, and the like).

[0259] The antisense oligonucleotide against the gene encoding theprotein of the present invention is useful as a medicament to treat orprevent diseases characterized by undesirable activation of NF-κ B, suchas inflammation, autoimmune diseases, infectious diseases (e.g., HIVinfection) and cancers. Thus, the present invention also includes apharmaceutical composition which comprises the above antisenseoligonucleotide as an active ingredient. The antisense oligonucleotidecan also be used to detect such diseases using northern hybridization orPCR.

[0260] The present invention also includes a ribozyme or a deoxyribozymewhich inhibits NF-κ B activation. A ribozyme or a deoxyribozyme is anRNA capable of recognizing a nucleotide sequence of a nucleic acid andcleaving the nucleic acid (see e.g., Hiroshi Yanagawa, “Jikken Igaku(Experimental Medicine) Bioscience 12: New Age of RNA). The ribozyme orthe deoxyribozyme can be produced so that it cleaves the selected targetRNA (e.g., mRNA encoding the protein of the present invention). Based onthe nucleotide sequence of the DNA encoding the protein of the presentinvention, the ribozyme or the deoxyribozyme specifically cleaving themRNA of the protein of the present invention can be designed. Suchribozyme or deoxyribozyme has a complementary sequence to the mRNA forthe protein of the present invention, complementarily associates withthe mRNA and then cleaves the mRNA, which results in reduction or entireloss of the expression of the protein of the present invention. Thelevel of the reduction of the expression is dependent on the level ofthe ribozyme or the deoxyribozyme expression in the target cells.

[0261] There are two types of ribozyme or deoxyribozyme commonly used: ahammerhead ribozyme or deoxyribozyme and a hairpin ribozyme ordeoxyribozyme. In particular, hammerhead ribozymes or deoxyribozymeshave been well studied regarding their primary and secondary structurenecessary for their cleavage activity, and those skilled in the art caneasily design the ribozymes or the deoxyribozymes nucleotided solely onthe nucleotide sequence information for the DNA encoding the protein ofthe present invention [see e.g., Iida et al., Saibou Kougaku Vol.16,No.3, p.438-445 (1997); Ohkawa & Taira, Jikken Igaku (ExperimentalMedicine) Vol.12, No.12, p.83-88 (1994)]. It is known that thehammerhead ribozymes or deoxyribozymes have a structure consisting oftwo recognition sites (recognition site I and recognition site IIforming a chain complementary to target RNA) and an active site, andcleave the target RNA at the 3′ end of its sequence NUX (wherein N is Aor G or C or U, and X is A or C or U)after the formation of acomplementary pair with the target RNA in the recognition sites. Inparticular, the sequence GUC (or GUA) has been found to have the highestactivity [see e.g., Koizumi, M. et al., Nucl. Acids Res. 17:7059-7071(1989); Iida et al., Saibou Kougaku Vol.16, No.3, p.438-445 (1997);Ohkawa & Taira, Jikken Igaku (Experimental Medicine) Vol.12, No.12,p.83-88 (1994); Kawasaki & Taira, Jikken Igaku (Experimental Medicine)Vol.18, No.3, p.381-386 (2000)].

[0262] Therefore the sequence GTC (or GTA) is searched out, and aribozyme is designed to form several,up to 10 to 20 complementary basepairs around that sequence. The suitability of the designed ribozyme orthe designed deoxyribozyme can be evaluated by checking whether theprepared ribozyme or the prepared deoxyribozyme can cleave the targetmRNA in vitro according to the method described for example in Ohkawa &Taira, Jikken Igaku (Experimental Medicine) Vol.12, No.12, p.83-88(1994). The ribozyme or the deoxyribozyme can be prepared by methodsknown in the art to synthesize RNA molecules.

[0263] Alternatively, the sequence of the ribozyme or the deoxyribozymecan be synthesized on a DNA synthesizer and inserted into variousvectors containing a suitable RNA polymerase promoter (e.g., T7 or SP6)to enzymatically synthesize an RNA molecule in vitro. Such ribozymes ordeoxyribozymes can be introduced into cells by gene transfer methodssuch as microinjection. Another method involves inserting a DNA encodingribozyme into a suitable expression vector and introducing the vectorinto cell strains, cells or tissues. Suitable vectors can be used tointroduce the ribozyme or the deoxyribozyme into a selected cell.Examples of vectors commonly used for such purpose include plasmidvectors and animal virus vectors (e.g., retrovirus, adenovirus, herpesor vaccinia virus vectors). Such ribozymes or deoxyribozymes are capableof inhibiting the NF-κ B activation mediated by the protein of thepresent invention.

[0264] According to the present invention, there is provided a doublestranded nucleic acid which inhibits an action of activating NF-κ B.That is, there is provided a double stranded nucleic acid whichpossesses a sequence corresponding to part of the nucleotide sequencesdescribed in (3) to (7) above. The term “a sequence corresponding topart of the nucleotide sequences” herein means that the sequence issubstantially identical to the part of the nucleotide sequences. Thatis, one strand of a double strand nucleic acid comprises a substantiallyidentical sequence with a part of the nucleotide sequences of interest,and the other strand of the double strand nucleic acid comprises asequence complementary to the above strand. The term “substantiallyidentical” means that the sequence is completely identical or identicalto a extent such that the double strand nucleic acid has an inhibitoryaction described below.

[0265] It has recently been clarified that when double stranded RNA(dsRNA) is introduced into a cell, a phenomenon occurs, which is knownas RNA interference (RNAi) wherein gene expression is inhibited as aresult of specific degradation of mRNA corresponding to that sequence(e.g. Fire A et al., Nature 391: p806-811 (1998), Elbashir S. M. et al.,Genes Dev. 15: p188-200 (2001)). dsRNA introduced into a cell isfragmentized into short interfering RNAs (siRNA) of 21-25 base pairs,due to an RNase specific for double stranded RNA, known as a Dicer,which belongs the RNase III family. This siRNA binds specifically with aprotein, to form a complex called as a RISC (RNA-induced silencingcomplex). This complex recognizes and binds with mRNA having the samesequence as the siRNA, cleaves target mRNA by a RNaseIII-like enzymeactivity at the center portion of siRNA, and as a result, geneexpression is inhibited. (Sharp P. A. Genes Dev.15: p485-490 (2001)).

[0266] The nucleotide sequence of the present invention can be used forpreparation of a double stranded nucleic acid which inhibits productionof the polypeptide of the present invention due to an RNA interferenceeffect against mRNA transcribed from the polynucleotide of the presentinvention. That is, the double stranded nucleic acid of the presentinvention is designed using the nucleotide sequence of thepolynucleotide of the present invention. When the double strandednucleic acid of the present invention is designed by the methoddescribed below, with reference to, for example, the reports of KazunoriTaira, et al.: RNAi Jikken Protocol, Yodosha (2003), and Elbashir S. M.et al.: Genes Dev. 15: p188-200 (2001), it can be obtained with higherprobability compared to where it is designed randomly: A regiondownstream of an initiation codon is selected, and from the selectedregion, a region consisting AA(N19-29)TT or AA(N21-31) is searched for,and the GC content of this sequence is calculated. A GC content of 50%is ideal; however, a sequence having a GC content of from at least 30%to 70% is selected. The sequence selected using the above criteria ischecked to determine if it is specific for the target gene by a BLAST(e.g. EST database of NCBI) search. A double stranded nucleic aciddesigned in this manner does not necessarily possess the desired RNAinterference effect. Evaluation of whether or not interference effect isexhibited can be performed by a method of confirming expressioninhibition effect wherein, using a suitable cell, a double strandednucleic acid is introduced or a double stranded nucleic acid isexpressed within the cell, and an amount of subject mRNA is measured(e.g. Northern blot or RT-PCR methods) or an amount of subject proteinis measured (e.g. Western blot or fluorescent antibody method) oractivity of the subject protein is measured, by methods known to personsskilled in the art.

[0267] The double stranded nucleic acid of the present inventionconsists of an antisense strand and a sense strand thereof. Theantisense strand comprises an antisense sequence of 18 to 29, preferably19 to 25 nucleotides, completely complementary to a partial sequence ofthe oligonucleotide of the present invention, and further, comprises 1to 4 bases at the 3′-end which protrude when annealed with the sensestrand (overhang). The sense strand ordinarily comprises a completelycomplementary sequence to the antisense strand, and further, comprises 1to 4 bases protruding at the 3′ end (overhang). To the extent that theantisense strand and the sense strand form a double strand, one or moremutations may be present in the sense strand. The nucleic acid of thesense strand and the antisense strand may be RNA, DNA, or a mixturethereof. However, it is preferable that the antisense sequence is RNA.Further, it is extremely preferable that both the sense strand and theantisense strand are RNA. The overhang portion may be formed withdeoxyribonucleotides G, A, T, and C and/or ribonucleotides G, A, U, andC, but a deoxyribonucleotide T and a ribonucleotide U are preferable.The number of overhang nucleotides is preferably 2 or 3, and 2, isextremely preferable. Suitable examples include UU (RNA) and TT (DNA).

[0268] Methods for preparing the double stranded nucleic acid of thepresent invention include chemical synthesis, methods of in vitrosynthesis and methods of effecting expression within a cell using anexpression vector (e.g. Takashi Morita, et al: Tanpakushitu KakusanKouso (Proteins, Nucleic Acids and Enzymes) Vol. 47 No. 14 p1939-1945(2002); Asako Sugimoto, Kagaku to Seibutsu (Chemistry and Biology) Vol.40 No. 11: p713-718 (2002); Makoto Miyagishi, et al.: Jikken Igaku(Experimental Medicine) Vol. 20 No. 18 p2667-2672 (2002); KazunoriTaira, et al.: RNAi Jikken Protocol, Yodosha (2003)).

[0269] A chemical synthesis method is a method where double strandednucleic acid is prepared by annealing an artificially synthesized sensestrand and antisense strand. The thus prepared double stranded nucleicacid can be introduced into a cell using a reagent such as FuGENE6(Roche) or Lipofectamine 2000 (Invitrogen).

[0270] A method of in vitro synthesis is a method of preparing a doublestranded nucleic acid (siRNA) wherein, for example, using T7 promoterand T7 RNA polymerase, a synthetic oligonucleotide having a 19-29 basesequence of the target gene is ligated downstream of the binding site ofT7 RNA polymerase, sense RNA and antisense RNA are synthesized by invitro transcription, and they are annealed in vitro. The prepared siRNAcan be introduced into a cell by lipofection methods using FuGENE6(Roche).

[0271] A method for effecting intracellular expression using anexpression vector is a method of effecting intracellular production of adouble stranded nucleic acid (siRNA) using an siRNA expression vector.Methods of intracellular synthesis of siRNA include, in addition to themethod described in the Examples, wherein a sense strand and anantisense strand are simultaneously expressed from both ends by twokinds of promoters, a method of, for example, effecting expression of asense strand and an antisense strand from separate transcription units,and a method of effecting expression of siRNA precursors which adopt ahairpin structure. As an expression vector, for example, pSilencer siRNAExpression Vector (Ambion Inc.) can be used.

[0272] Below, as a specific example, a double stranded nucleic acidpossessing an expression inhibiting effect due to RNA interferenceagainst expression of the polypeptide represented by SEQ ID NO: 87,which is encoded by the polynucleotide of SEQ ID NO: 88, is discussed.

[0273] First, after synthesizing each oligonucleotide pairs described in(A) to (F), they were respectively annealed and double stranded nucleicacids were obtained as sequences corresponding to a part of thenucleotide sequence of SEQ ID NO: 88.

[0274] (A) sense strand (A1) 5′-GUCCAGGAUAUCAUGAGUCTT-3′ (SEQ ID NO:185) antisense strand (B1) 3′-TTCAGGUCCUAUAGUACUCAG-5′ (SEQ ID NO: 186)

[0275] (B) sense strand (A2) 5′-GAAGUCUGAAGAUCUAUCCTT-3′ (SEQ ID NO:187) antisense strand (B2) 3′-TTCUUCAGACUUCUAGAUAGG-5′ (SEQ ID NO: 188)

[0276] (C) sense strand (A3) 5′-GCUGAAGAAGAGGUGUUCCTT-3′ (SEQ ID NO:189) antisense strand (B3) 3′-TTCGACUUCUUCUCCACAAGG-5′ (SEQ ID NO: 190)

[0277] (D) sense strand (A4) 5′-GAUGACACAGAUGAAGCCCTT-3′ (SEQ ID NO:191) antisense strand (B4) 3′-TTCUACUGUGUCUACUUCGGG-5′ (SEQ ID NO: 192)

[0278] (E) sense strand (A5) 5′-GCCCUCAGAGUCCAGAAUCTT-3′ (SEQ ID NO:193) antisense strand (B5) 3′-TTCGGGAGUCUCAGGUCUUAG-5′ (SEQ ID NO: 194)

[0279] (F) sense strand (A6) 5′-GAUGACUUUGGUAUCAAACTT-3′ (SEQ ID NO:195) antisense strand (B6) 3′-TTCUACUGAAACCAUAGUUUG-5′ (SEQ ID NO: 196)

[0280] (in oligonucleotides (A) to (F) above, A, U, G and C representeach ribonucleotide, respectively, and T representsdeoxyribonucleotide.)

[0281] The above oligonucleotide pairs (A) to (F) are preferablenon-limiting examples of oligonucleotide pairs (a) to (f) in above item(25).

[0282] Using EBNA cells (Invitrogen), the RNA interference effectspossessed by the double stranded nucleic acids prepared from (A) to (F)above were confirmed. That is, using Lipofectamine 2000 (Invitrogen), anexpression vector having the polynucleotide of SEQ ID NO:88, pNFkB-Luc,phRL-TK vector and double stranded nucleic acid (A) were co-transfectedinto EBNA cells, and after culturing, luciferase activity was measured.As a result, it was found that the activity of cells into which doublestranded nucleic acid (A) had been co-transfected was markedly lower incomparison with the activity of cells into which double stranded nucleicacid had not been introduced. This result indicated that the doublestranded nucleic acid (A) markedly inhibited the expression of theprotein represented by SEQ ID NO: 87.

[0283] Also, in the case where the double stranded nucleic acid of (B),(C), (D), (E) or (F) was co-transfected, it was found that expression ofthe protein represented by SEQ ID NO: 87, was similarly inhibited.

[0284] Therefore, because the double stranded nucleic acids of (A) to(F) above possessed an effect of inhibiting expression of the proteinrepresented by SEQ ID NO:87, they are useful as expression inhibitingagents in respect of the protein represented by SEQ ID NO: 87.

[0285] Next, an expression vector (pUH1) to enable intracellularexpression of siRNA was prepared. (G) 5′-AAAAGTCCAGGATATCATGAGTCTTTTTTA(SEQ ID NO:197) 5′-AGCTTAAAAAAGACTCATGATATCCTGGAC (SEQ ID NO:198) (H)5′-AAAAGAAGTCTGAAGATCTATCCTTTTTTA (SEQ ID NO:199)5′-AGCTTAAAAAAGGATAGATCTTCAGACTTC (SEQ ID NO:200) (I)5′-AAAAGCTGAAGAAGAGGTGTTCCTTTTTTA (SEQ ID NO:201)5′-AGCTTAAAAAAGGAACACCTCTTCTTCAGC (SEQ ID NO:202) (J)5′-AAAAGATGACACAGATGAAGCCCTTTTTTA (SEQ ID NO:203)5′-AGCTTAAAAAAGGGCTTCATCTGTGTCATC (SEQ ID NO:204) (K)5′-AAAAGCCCTCAGAGTCCAGAATCTTTTTTA (SEQ ID NO:205)5′-AGCTTAAAAAAGATTCTGGACTCTGAGGGC (SEQ ID NO:206) (L)5′-AAAAGATGACTTTGGTATCAAACTTTTTTA (SEQ ID NO:207)5′-AGCTTAAAAAAGTTTGATACCAAAGTCATC (SEQ ID NO:208)

[0286] The oligonucleotide pairs of (G) to (L) above were thensynthesized. Thereafter, each oligonucleotide pair were respectivelyannealed to obtain double stranded DNA, and expression vectors forexpression of siRNA (pUH88-1, pUH88-2, pUH88-3, pUH88-4, pUH88-5 andpUH88-6) were prepared.

[0287] The RNA interference effect possessed by the expression vectorsfor expression of siRNA (pUH88-1, pUH88-2, pUH88-3, pUH88-4, pUH88-5 andpUH88-6) was confirmed using EBNA cells (Invitrogen). That is to say, anexpression vector having a polynucleotide of SEQ ID NO: 88, pNFkB-Luc,phRL-TK vector and pUH88-1 were co-transfected into EBNA cells usingFuGENE6, and after culturing luciferase activity was measured. As aresult, it was found that the activity of a cell into which pUH88-1 wasco-transfected was markedly low in comparison to the activity of a cellinto which pUH1 was co-transfected, which was used as a control. Thisresult indicated that siRNA, expressed intracellularly by pUH88-1,markedly inhibited expression of the protein represented by SEQ ID NO:87.

[0288] In cases where pUH88-2, pUH88-3, pUH88-4, pUH88-5 or pUH88-6 wasco-transfected, it was found that expression of the protein representedby SEQ ID NO: 87 was similarly markedly inhibited.

[0289] Therefore, because the expression vector for expression of theabove described siRNA, has an effect of inhibiting expression of theprotein represented by SEQ ID NO: 87, it is useful as an expressioninhibiting agent in respect of the protein represented by SEQ ID NO:87.

[0290] Further, the present invention encompasses double strandednucleic acids, and vectors expressing these double stranded nucleicacids, which have activity to inhibit expression of the protein of SEQID NO: 87 and which are formed by annealing of an antisense strandhaving a sequence including an antisense sequence of a antisense stranddescribed in (A) to (F) above with 1 to 4 bases at the 3′ end whichprotrude when annealed with a sense strand (overhang); and a sensestrand having a sequence including a sense sequence of a sense stranddescribed in (A) to (F) above with 1 to 4 bases at the 3′ end whichprotrude when annealed with an antisense strand (overhang).

[0291] The double stranded nucleic acids of the present invention forthe polynucleotide of the present invention, and the vectors whichexpress these double stranded nucleic acids are useful as expressioninhibiting agents in respect of the polynucleotide of the presentinvention. Further, they are useful as a medicament for the treatment orprevention of diseases characterized by undesirable activation of NF-κ Bsuch as inflammation, auto-immune disease, infection, cancer, etc. Thepresent invention also encompasses a pharmaceutical compositioncomprising the above double stranded nucleic acids and/or the aboveexpression vectors, as an active ingredient.

[0292] The double-stranded nucleic acid of the present invention can beused to examine at what position the polypeptide encoded by the gene ofthe present invention functions in the signal transduction pathway whichbrings about NF-κB activation. Specific examples include theidentification of the functioning position of the polypeptide encoded bythe gene of the present invention represented by SEQ ID NO: 88 by usingthe double-stranded nucleic acid of the present invention prepared usingoligonucleotides having nucleotide sequences represented by SEQ ID NOs:185-196.

[0293] From a different viewpoint, a polypeptide having an action ofactivating NF-κB encoded by a gene of the present invention whosefunctioning position has been identified using the double-strandednucleic acid of the present invention, and the gene of the presentinvention, are useful, for example, as targets for new drug developmentand/or as aides to developmental research. Specific examples include apolypeptide encoded by the gene of the present invention represented bySEQ ID NO: 88 whose functioning position has been identified by thedouble-stranded nucleic acid of the present invention prepared usingoligonucleotides having nucleotide sequences represented by SEQ ID NOs:185-196.

[0294] The above described experiment can be conducted, for example, inthe following manner. A double-stranded nucleic acid preparedcorresponding to a gene of the present invention is introduced into asuitable cell, and the cell is subjected to various suitable stimuliwhich activate NF-κB, and the observed pattern of inhibition of NF-κBactivation is analyzed in detail, and the position at which thepolypeptide encoded by the gene of SEQ ID NO: 88 is identified. Examplesof suitable cells include HEK293 (ATCC CRL1573), HeLa (ATCC CCL2.2),MRC-5 (ATCC CCL171), THP-1 (ATCC TIB-202), RAW264.7 (ATCC TIB-71),Normal Human lung fibroblast (Cryo NHLF: Sanko Junyaku Co. Ltd.) and thelike. Examples of various suitable stimuli include those induced byIL-1, TNF, lipopolysaccharide, double-stranded RNA, bacteriallipopeptides, unmethylated CpG DNA and the like.

[0295] The present invention, moreover, relates to a process forobtaining a new gene having a function, which comprises using theoligo-capping method to construct a full-length cDNA library, and usinga signal factor indicative of the presence of a protein having thefunction. An example of such signal factor is a reporter gene.

[0296] Methods using a cDNA library containing a lot of non-full-lengthcDNAs are inefficient in obtaining many genes (cDNAs) having functions.Therefore libraries with a high ratio of the number of the full-lengthcDNA clones to the total number of the clones are necessary.“Full-length cDNA” refers to a complete DNA copy of mRNA from a gene.The cDNA libraries produced using the oligo-capping method containfull-length cDNA clones in a ratio of 50 to 80%, namely, a 5 to 10-foldincrease in full-length cDNA clones compared to the cDNA librariesproduced by prior art methods (Sumio Sugano, the monthly magazine BIOINDUSTRY Vol.16, No.11, p.19-26). Full-length cDNA clones are essentialfor protein expression in functional analyses of genes, and full-lengthcDNA clones themselves are very important materials for activitymeasurement. Thus, cloning of full-length cDNA is necessary forfunctional analyses of genes. Sequencing of the cDNA not only providesimportant information for establishing the primary sequence of theprotein encoded by the cDNA, but also reveals the entire exon sequence.Thus, the full-length cDNA provides valuable information for identifyinga gene, such as information for determining the primary sequence of aprotein, exon-intron structure, the transcription initiation site ofmRNA, the location of a promoter, etc.

[0297] The construction of full-length cDNA libraries by theoligo-capping method can be carried out, for example, according to themethod described in “Shin Idenshi Kougaku Handbook (New GeneticEngineering Handbook)”, the third edition (1999), an extra issue of“Jikken Igaku (Experimental Medicine)”, YODOSHA CO., LTD. The reportergene indicative of the presence of a protein having a function containsone or more suitable expression regulation sequence portion to which aprotein factor such as a transcriptional factor can bind, and astructural gene portion which allows the measurement of the activationof the proteins factor. The structural gene portion may encode anypeptide or protein so long as those skilled in the art can measure theactivity or amount of its expression product (including the amount ofthe mRNA produced). For example, chloramphenicol acetyltransferase,β-galactosidase, luciferase, etc., can be used and their enzymaticactivity measured.

[0298] The oligo-capping method used herein involves substituting a capstructure with a synthetic oligo sequence by using BAP, TAP and an RNAligase, as described in Suzuki & Sugano, “Shin Idenshi Kougaku Handbook(New Genetic Engineering Handbook)”, the third edition (1999), an extraissue of “Jikken Igaku (Experimental Medicine)”, YODOSHA CO., LTD.

[0299] The process of the present invention uses an in vitro system or acell-based system, preferably a cell-based system. Examples of suchcells include cells of prokaryotes such as E. coli, microorganisms suchas yeast and fungi, as well as insects and animals. Preferred examplesinclude animal cells, in particular, 293-EBNA cells and NIH3T3 cells.

[0300] Examples of reporter genes indicative of the presence of aprotein having a function include reporter genes containing a CREB (cAMPresponsive element binding protein) binding sequence or AP-1 (activatorprotein-1) binding sequence at the expression regulation sequence regionof the reporter genes, in addition to the NF-κ B reporter genesdescribed herein. For example, if a gene capable of activating CREB isto be obtained, a CREB-dependent reporter plasmid and an expressionvector comprising full-length cDNA produced by the oligo-capping methodcan be cotransfected into cells, and an expression vector havingincreased reporter activity can be selected from the cells to attain thepurpose. If a gene capable of inhibiting CREB is to be obtained, aCREB-dependent reporter plasmid and an expression vector comprisingfull-length cDNA produced by the oligo-capping method can becotransfected into cells, and an expression vector having decreasedreporter activity can be selected from the cells to attain the purpose.These procedures may be carried out in the presence of a certainstimulus to the cells. The cDNA clone (expression vector) to betransfected into the cells may be a single clone or multiple cloneswhich may be transfected simultaneously. One embodiment of the processof the present invention is detailed in Examples herein. Alternatively,a screening system for obtaining a gene capable of inhibiting NF-κ Bactivation can also be constructed by cotransfecting an expressionvector comprising full-length cDNA and a reporter gene into cells,stimulating the cells with IL-1 or TNF-α and the like, and selecting aclone having subnormally increased reporter activity.

[0301] However, the process of the present invention is not limited tothese embodiments.

[0302] Because the cDNA of the present invention is full-length, its 5′end sequence is the transcription initiation site of the correspondingmRNA. Therefore the cDNA sequence can be used to identify the promoterregion of the gene by comparing the cDNA with the genomic nucleotidesequence. Genomic nucleotide sequences are available from variousdatabases when the sequences have been deposited in the databases.Alternatively, the cDNA can also be used to clone the desired sequencefrom a genomic library, for example, by hybridization, and determine itsnucleotide sequence. Thus, by comparing the nucleotide sequence of thecDNA of the present invention with a genomic sequence, the promoterregion of the gene located upstream the cDNA can be identified. Inaddition, the promoter fragment thus identified can be used to constructa reporter plasmid for evaluating the expression of the gene. Ingeneral, the DNA fragment spanning 2 kb (preferably 1 kb) upstream fromthe transcription initiation site can be inserted upstream of thereporter gene to produce the reporter plasmid. The reporter plasmid canbe used to screen for a compound which enhances or reduces theexpression of the gene. For example, such screening can be carried outby transforming a suitable cell with the reporter plasmid, culturing thetransformed cell for a certain period of time, adding a certain amountof a test compound, measuring the reporter activity expressed by thecell after a certain period of time, and comparing the activity withthat of a cell to which the test compound has not been added. Thesemethods are also included in the scope of the present invention.

[0303] The present invention also relates to a computer-readable mediumon which a sequence data set has been stored, said sequence data setcomprising at least one nucleotide sequence selected from the groupconsisting of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24,26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60,62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96,98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124,126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 151,153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173, 175, 177 and 179,and/or at least one amino acid sequence selected from the groupconsisting of SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25,27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61,63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97,99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125,127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 152, 154,156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178 and 180.

[0304] In another aspect, the present invention relates to a method forcalculating a homology, which comprises comparing data on the abovemedium with data of other nucleotide sequences. Thus, the gene and aminoacid sequence of the present invention provide valuable information fordetermining their secondary and tertiary structure, e.g., informationfor identifying other sequence having a similar function and highhomology. These sequences are stored on the computer-readable medium,then a database is searched using data stored in a known macromoleculestructure program and a known search tool such as GCG. In this manner, asequence in a database having a certain homology can be easily found.

[0305] The computer-readable medium may be any composition of materialsused to store information or data. Examples of such media includecommercially available floppy disks, tapes, chips, hard drives, compactdisks and video disks. The data on the medium allows a method forcalculating a homology by comparing the data with other nucleotidesequence data. This method comprises the steps of providing a firstpolynucleotide sequence containing the polynucleotide sequence of thepresent invention for the computer-readable medium, and then comparingthe first polynucleotide sequence with at least one-secondpolynucleotide or polypeptide sequence to identify the homology.

[0306] The present invention also relates to an insoluble substrate towhich polynucleotide comprising all or part of the nucleotide sequencesselected from the group consisting of SEQ ID NOS: 2, 4, 6, 8, 10, 12,14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48,50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84,86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116,118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144,146, 148, 150, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171,173, 175, 177 and 179, are fixed. A plurality of the variouspolynucleotides which are DNA probes are fixed on a specificallyprocessed solid substrate such as slide glass to form a DNA microarrayand then a labeled target polynucleotide is hybridized with the fixedpolynucleotides to detect a signal from each of the probes. The dataobtained is analyzed and the gene expression is determined.

[0307] The present invention further relates to an insoluble substrateto which polypeptides comprising all or part of the amino acid sequencesselected from the group consisting of SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13,15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49,51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85,87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117,119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145,147, 149, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174,176, 178 and 180, are fixed. By mixing organism-derived cell extractwith the insoluble substrate on which these proteins are fixed, it ispossible to isolate or identify cell-derived components such as proteinscaptured on the insoluble substrate that can be expected to be useful indiagnosis or drug development.

EXAMPLES

[0308] The following examples further illustrate, but do not limit thepresent invention.

Example 1

[0309] Construction of a Full-length cDNA Library Using theOligo-capping Method

[0310] (1) Preparation of RNA from Human Lung Fibroblasts (Cryo NHLF)

[0311] Human lung fibroblasts (Cryo NHLF: purchased from Sanko JunyakuCo., Ltd.) were cultured according to the attached protocol. Afterrepeating subculturing the cells to obtain fifty 10 cm dishes containingthe resulting culture, the cells were recovered with a cell scraper.Then, total RNA was obtained from the recovered cells by using the RNAextraction reagent ISOGEN (purchased from NIPPON GENE) according to themanufacturer's protocol. Then, poly A⁺ RNA was obtained from the totalRNA by using an oligo-dT cellulose column according to Maniatis et al.,supra.

[0312] (2) Preparation of RNA from Mouse ATDC5 Cells

[0313] ATDC5, a cell strain cloned from mouse EC (embryonal carcinoma)(Atsumi, T. et al.: Cell Diff. Dev., 30: p109-116)(1990) was repeatedlysubcultured to obtain fifty 10 cm dishes containing the resultantculture. Thereafter, poly A⁺ RNA was obtained by a method similar tothat of (1) above. Culture of ATDC5 cells was performed according to themethod described in Atsumi, T. et al.: Cell Diff. Dev., 30: p109-116(1990).

[0314] (3) Construction of a Full-length cDNA Library by theOligo-capping Method

[0315] A full-length cDNA library was constructed from poly A⁺ RNA ofthe above human lung fibroblasts and ATDC5 cells by the oligo-cappingmethod according to the method of Sugano S. et al. [e.g., Maruyama, K. &Sugano, S., Gene, 138:171-174 (1994); Suzuki, Y. et al., Gene,200:149-156 (1997); Suzuki, Y. & Sugano, S. “Shin Idenshi KougakuHandbook (New Genetic Engineering Handbook)”, the third edition (1999),an extra issue of “Jikken Igaku (Experimental Medicine)”, YODOSHA CO.,LTD.].

[0316] (4) Preparation of Plasmid DNA

[0317] The full-length cDNA library constructed as above was transfectedinto E. coli strain TOP 10 by electroporation, then spread on LB agarmedium containing 100 μg/ml of ampicillin, and incubated overnight at37° C. Then, using QIAwell 96 Ultra Plasmid Kit (QIAGEN) according tothe manufacturer's protocol, the plasmids were recovered from thecolonies grown on ampicillin-containing LB agar medium.

Example 2

[0318] Cloning of DNA Capable of Activating NF-κ B

[0319] (1) Screening of the cDNA Encoding the Protein Capable ofActivating NF-κ B

[0320] 293-EBNA cells (purchased from Invitrogen) were grown to 1×10⁴cells/100 μl/well in a 96 well plate for cell culture for 24 hours at37° C. (in the presence of 5% CO₂) using 5% FBS containing DMEM medium.Then, 50 ng of pNF κ B-Luc (purchased from STRATAGENE) and 2 μl of thefull-length cDNA expression vector prepared in above Example 1.(4) werecotransfected into the cells in a well using FuGENE 6 (purchased fromRoche) according to the manufacturer's protocol. After 24 hours ofculture at 37° C., the reporter activity of NF-κ B (luciferase activity)was measured using long-term luciferase assay system,PIKKA GENE LT2.0(TOYO INK) according to the attached manufacturer's instructions. Theluciferase activity was measured using Wallac ARVO™ST 1420 MULTILABELCOUNTER (Perkin Elmer).

[0321] (2) DNA Sequencing

[0322] The above screening was carried out for 155,000 clones, andplasmids showing a 5-fold or more increase in luciferase activitycompared to that of the control experiment (luciferase activity of thecell into which vacant vector pME18S-FL3 is introduced instead offull-length cDNA expression vector) were selected. One pass sequencingwas carried out from the 5′ end of the cloned cDNA (sequencing primer:5′-CTTCTGCTCTAAAAGCTGCG-3′ (SEQ ID NO: 181)) and from the 3′ end(sequencing primer: 5′-CGACCTGCAGCTCGAGCACA-3′ (SEQ ID NO: 182)) so thatas long sequence as possible is determined. The sequencing was carriedout using the reagent Thermo Sequenase II Dye Terminator CycleSequencing Kit (Amersham Pharmacia Biotech) or BigDye Terminator CycleSequencing FS Ready Reaction Kit (Applied Biosystems) and the device ABIPRISM 377 sequencer or ABI PRISM 3100 sequencer according to themanufacturer's instructions.

[0323] (3) Database Analysis of the Obtained Clones

[0324] BLAST (Basic local alignment search tool) searching [S. F.Altschul et al., J. Mol. Biol., 215:403-410 (1990)] was carried out inGenBank for the obtained nucleotide sequence. The results showed that148 clones represented 90 genes encoding new proteins capable ofactivating NF-κ B.

[0325] (4) Full-length Sequencing

[0326] The full-length DNA sequences for the 90 new clones weredetermined (SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26,28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62,64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98,100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126,128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 151, 153,155, 157, 159, 161, 163, 165, 167, 169, 171, 173, 175, 177 and 179). Theamino acid sequences of the protein coding regions (open reading frames)were deduced (SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25,27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61,63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97,99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125,127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 152, 154,156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178 and 180).

Example 3

[0327] Screening Compounds Inhibiting NF-κ B Activation

[0328] 293-EBNA cells were seeded on 5% FBS containing DMEM medium in a96-well cell culture plate to a final cell density of 1×10⁴ cells/100μl/well, and cultured for 24 hours at 37° C. in the presence of 5% CO₂.Then, 50 ng of the expression vector comprising the gene encoding NF-κ Bactivating protein of SEQ ID NO: 5, 9, 17, 21, 35, 37, 41, 53, 57, 63,67, 71, 75, 81, 87, 91, 93, 97, 121, 123, 129, 154, 158, 162, 168, 170,172, 176 or 178, and 50 ng of the reporter plasmid pNF κB-Luc werecotransfected into the cells in a well using FuGENE 6. After 1 hour, theproteasome inhibitor MG-132 (purchased form CALBIOCHEM) (Uehara T. etal., J. Biol. Chem. 274, p.15875-15882 (1999); Wang X. C. et al.,Invest. Ophathalmol. Vis. Sci. 40, p.477-486) was added to the cultureto final concentrations of 0.1 μM, 0.5 μM, 1.0 μM and 10 μM,respectively. After 24 hours of culture at 37° C., the reporter activitywas measured using PIKKA GENE LT2.0. The results showed that MG132inhibited the expression of the reporter gene (FIG. 1 to FIG. 29).

Example 4

[0329] Intracellular Localization

[0330] (1) Preparation of Expression Vector pDEST-NGFP for Adding GFP tothe N-terminus of the Target Protein

[0331] After cleaving pQBI25-fC1 (purchased from Wako Pure ChemicalIndustries, Ltd.) with restriction enzyme Apa I, the ends thereof wereblunted with a DNA Blunting Kit (manufactured by Takara Bio Inc.) andblunted fragment was then linked with rfC cassette of Gateway VectorConversion System (Invitrogen) with T4 DNA ligase to prepare pDEST-NGFP.

[0332] (2) Preparation of Expression Vector pDEST-CGFP for Adding GFP tothe C-terminus of the Target Protein

[0333] After cleaving pQBI25-fA1 (purchased from Wako Pure ChemicalIndustries, Ltd.) with restriction enzyme Bg1 II, the ends thereof wereblunted with a DNA Blunting Kit (manufactured by Takara Bio Inc.) andblunted fragments were subjected to self-ligation. After transformingE.coli DH5 strain, plasmids were prepared by standard methods. Next, theplasmid was cleaved with restriction enzymes Nru I and Sac II, and afterblunting the ends thereof, the fragment was linked to rfB cassette ofGateway Vector Conversion System (Invitrogen) to prepare pDEST-CGFP.

[0334] (3) Intracellular Localization of the Protein (SEQ ID NO: 5)Encoded by the Polynucleotide of SEQ ID NO: 6.

[0335] Using the expression vectors prepared in (1) and (2) above,pDEST-NGFP and pDEST-CGFP, expression vectors, pDEST-NGFP 5 andpDEST-CGFP 5, for expressing a fusion protein of the protein (SEQ ID NO:5) encoded by the polynucleotide of SEQ ID NO: 6 and GFP, wererespectively prepared. Preparation of expression vectors was performedwith Gateway Technology (Invitrogen), in accordance with theirrecommended protocol.

[0336] Africa Green Monkey kidney-derived Vero cells (obtained fromATCC) were inoculated on Opti-MEM medium (Invitrogen) containing 5% FBSin a 96-well EZView culture plate (manufactured by Asahi TechnoglassCorporation) to a cell density of 5000 cells/100 μl/well, and culturedfor 24 hours at 37° C. in the presence of 5% CO₂. Next, using FuGENE6,100 ng of the expression vector pDEST-NGFP5, or pDEST-CGFP5, was addedto 1 well. After culturing for 24 or 48 hours at 37° C., cells wereobserved with a fluorescence microscope. For observation with afluorescence microscope, IX70 manufactured by Olympus was used, andobservation was performed with the NIBA filter set of the microscope.Results showed localization of the protein of SEQ ID NO: 5 primarily inendoplasmic reticulum.

[0337] (4) Intracellular Localization of the Protein (SEQ ID NO: 87)Encoded by the Polynucleotide of SEQ ID NO: 88.

[0338] Using the expression vector pDEST-CGFP prepared in (2) above, anexpression vector, pDEST-CGFP87, for expressing a fusion protein of theprotein (SEQ ID NO: 87) encoded by the polynucleotide of SEQ ID NO: 88,and GFP, was prepared. Preparation of the expression vector wasperformed with Gateway Technology (Invitrogen), in accordance with theirrecommended protocol.

[0339] Africa green monkey kidney-derived Vero cells (obtained fromATCC) were inoculated on Opti-MEM medium (Invitrogen) containing 5% FBSin a 96-well EZView culture plate (manufactured by Asahi TechnoglassCorporation) to a cell density of 5000 cells/100 μl/well,_and culturedfor 24 hours at 37° C. in the presence of 5% CO₂. Then using FuGENE6,100 ng of the expression vector pDEST-CGFP87 was added to 1 well. Afterculturing for 24 or 48 hours at 37° C., cells were observed with afluorescence microscope. For observation with a fluorescence microscope,IX70 manufactured by Olympus was used, and observation was performedwith the NIBA filter set of the microscope. Results showed localizationof the protein of SEQ ID NO: 87 in cell membrane.

Example 5

[0340] Induction of Expression of Human Interferon (IFN)-β by Using theProtein (SEQ ID NO: 154) Encoded by the Polynucleotide of SEQ ID NO:153.

[0341] EBNA cells (Invitrogen) were inoculated on DMEM medium containing5% FBS in a 96-well cell culture plate to a cell density of 1.2×10⁴cells/100 μl/well, and cultured for 24 hours at 37° C. in the presenceof 5% CO₂. Then using FuGENE6, 20 ng of the expression vector having thenucleotide sequence of SEQ ID NO: 153 and 50 ng of a reporter plasmidhaving human interferon (IFN)-β gene promoter were added to 1 well.After 24 hours of culture at 37° C., the reporter activity (luciferaseactivity) was measured using PicaGene LT2.0. Results indicated thatcells into which the expression vector having the nucleotide sequence ofSEQ ID NO: 153 was introduced had luciferase activity 100 or more timesgreater than cells of a control experiment (luciferase activity of cellsinto which null vector pME18S-FL3 was introduced).

[0342] It should be noted that the reporter plasmid having human IFN-βgene promoter was prepared in the following method. Primers of twosynthetic oligonucleotides: (SEQ ID NO:183)5′-CTAGCTAGCTAGAAACTACTAAAATGTAAATGACATAG-3′ and (SEQ ID NO:184)5′-CGCAAGCTTGAAAGGTTGCAGTTAGAATGTCCTTTC-3′,

[0343] were designed, and using this primer pair, PCR was performedusing human genome (CLONTECH) as a template. An amplified fragment ofapprox. 0.15 kb was isolated, and after digesting with NheI and HindIIIrestriction enzymes, the fragment was inserted between the NheI site andHindIII site of firefly luciferase reporter vector pGL3-Basic Vector(Promega Corporation) using T4 DNA ligase to prepare the reporterplasmid. PCR was performed by preparing 50 μl of reaction solutioncontaining 1 μl of KOD-Plus-(TOYOBO), 5 μl of 10×PCR Buffer (suppliedwith KOD-Plus-), 5 μl of 2 mM dNTPs (supplied with KOD-Plus-), 2 μl of25 mM MgSO₄ (supplied with KOD-Plus-), 1.5 μl of each of the aboveprimers (each at a concentration of 10 μM) and 100 ng of human GenomicDNA (CLONTECH) and after 2 minutes incubation at 94° C., conducting 45cycles of incubation for 15 seconds at 94° C., 30 seconds at 60° C., and40 seconds at 68° C. using Takara PCR Thermal Cycler MP (manufactured byTakara Bio Inc.).

Example 6

[0344] Inhibition of Expression of the Gene of SEQ ID NO:88 by DoubleStranded Nucleic Acid.

[0345] (1) Preparation of Double Stranded Nucleic Acid

[0346] First, oligonucleotides having the nucleotide sequences describedbelow were prepared by ordinary chemical synthesis. It should be notedthat in the sequences described below, A, G, C and U represent eachribonucleotide, and T represents deoxyribonucleotide. A15′-GUCCAGGAUAUCAUGAGUCTT-3′ (SEQ ID NO:185) B15′-GACUCAUGAUAUCCUGGACTT-3′ (SEQ ID NO:186) A25′-GAAGUCUGAAGAUCUAUCCTT-3′ (SEQ ID NO:187) B25′-GGAUAGAUCUUCAGACUUCTT-3′ (SEQ ID NO:188) A35′-GCUGAAGAAGAGGUGUUCCTT-3′ (SEQ ID NO:189) B35′-GGAACACCUCUUCUUCAGCTT-3′ (SEQ ID NO:190) A45′-GAUGACACAGAUGAAGCCCTT-3′ (SEQ ID NO:191) B45′-GGGCUUCAUCUGUGUCAUCTT-3′ (SEQ ID NO:192) A55′-GCCCUCAGAGUCCAGAAUCTT-3′ (SEQ ID NO:193) B55′-GAUUCUGGACUCUGAGGGCTT-3′ (SEQ ID NO:194) A65′-GAUGACUUUGGUAUCAAACTT-3′ (SEQ ID NO:195) B65′-GUUUGAUACCAAAGUCAUCTT-3′ (SEQ ID NO:196)

[0347] Next, the oligonucleotides were each dissolved in RNase-freewater to a concentration of 50 μM (50 pmol/μl). Then, 30 μl each of thedissolved oligonucleotides A1 and B1, and 15 μl of 5×annealing buffer(500 mM potassium acetate, 150 mM HEPES-KOH pH 7.4, 10 mM magnesiumacetate) were mixed (total: 75 μl). After heating the solution for 1minute at 90° C., the solution was incubated for 60 minutes at 37° C.,and the two oligonucleotides were annealed to prepare a double strandednucleic acid. The double stranded nucleic acid was designated 88-1.

[0348] By the same method as described above, the oligonucleotides A2and B2 were annealed to prepare double stranded nucleic acid 88-2.

[0349] By the same method as described above, the oligonucleotides A3and B3 were annealed to prepare double stranded nucleic acid 88-3.

[0350] By the same method as described above, the oligonucleotides A4and B4 were annealed to prepare double stranded nucleic acid 88-4.

[0351] By the same method as described above, the oligonucleotides A5and B5 were annealed to prepare double stranded nucleic acid 88-5.

[0352] By the same method as described above, the oligonucleotides A6and B6 were annealed to prepare double stranded nucleic acid 88-6.

[0353] (2) Expression Inhibition of the Gene of SEQ ID NO:88 by DoubleStranded Nucleic Acid Prepared in (1) Above

[0354] EBNA cells (Invitrogen) were inoculated on DMEM medium containing5% FBS in a 96-well cell culture plate to a cell density of 1.2×10⁴cells/100 μl/well, and cultured for 24 hours at 37° C. in the presenceof 5% CO₂. Next, using Lipofectamine 2000 (Invitrogen), 50 ng of anexpression vector having the polynucleotide of SEQ ID NO:88 and 50 ng ofpNFkB-Luc (STRATAGENE) and 10 ng of phRL-TK vector (Promega Corporation)used as an internal standard, were co-transfected with double strandednucleic acid 88-1 (10 pmol) prepared in (1) above into 1 well. Afterculturing for 24 hours at 37° C., firefly luciferase activity andRenilla reniformis luciferase activity were measured using aDual-Luciferase Reporter Assay System (Promega Corporation). Further,each of 88-2 (10 pmol), 88-3 (10 pmol), 88-4 (10 pmol), 88-5 (10 pmol)or 88-6 (10 pmol) was co-transfected into 1 well in place of 88-1 (10pmol), and luciferase activity was measured in the same manner. Resultsare shown in Table 1. In Table 1, values for activity represent relativevalues when firefly luciferase activity in the control experiment (cellsinto which double stranded nucleic acid has not been introduced) isestablished as 100%. (It should be noted that values for fireflyluciferase activity were standardized with values for Renilla reniformisluciferase activity used as an internal standard, before using thesevalues in calculations.)

[0355] As shown in Table 1, cells into which double stranded nucleicacid 88-1, 88-2, 88-3, 88-4, 88-5 or 88-6 has been introduced exhibitedinhibition of firefly luciferase activity relative to the controlexperiment (cells into which double stranded nucleic acid has not beenintroduced). These results indicated that double stranded nucleic acids88-1, 88-2, 88-3, 88-4, 88-5 and 88-6 inhibited expression of the geneof SEQ ID NO: 88. TABLE 1 double stranded nucleic acid relativeluciferase activity (%) 88-1 18 88-2 12 88-3 9 88-4 8 88-5 5 88-6 7

Example 7

[0356] Expression Inhibition of the Gene of SEQ ID NO: 88 Using siRNAExpression Vector

[0357] (1) Preparation of siRNA Expression Vector pUH1

[0358] Expression vector pUH1, which is used in the preparation ofexpression vectors for expression of siRNA in animal cells, was preparedas follows.

[0359] First, in order to clone a promoter fragment of human U6 smallnuclear RNA (GenBank Accession Number: M14486) gene, the following twooligonucleotides primers: (SEQ ID NO:209)5′-GCGAATTCGGGCAGGAAGAGGGCCTATTTCCCAT-3′, and (SEQ ID NO:210)5′-GCAAGCTTTTTTGTCTTCTTTCCACAAGATATATAAAGCCAAG-3′

[0360] were synthesized, and using this primer pair, PCR was performedwith human genome (CLONTECH) as a template. An amplified fragment ofapprox. 0.27 kb was isolated, and after digestion with restrictionenzymes EcoRI and HindIII, the fragment was inserted between EcoRI andHindIII sites of pBluescript II KS(+) (STRATAGENE) using T4 DNA ligase.Thereafter, E.coli DH5 (TOYOBO) was transformed and the target plasmidwas obtained (and designated pU1).

[0361] Then, in order to clone a promoter fragment of human H1 RNA(GenBank Accession Number: X16612) gene, the following twooligonucleotide primers: (SEQ ID NO:211)5′-CGCTCGAGCCATGGAATTCGAACGCTGACGTC-3′, and (SEQ ID NO:212)5′-GCAAGCTTTCTCATACAGAACTTATAAGATTCCC-3′

[0362] were synthesized, and using this primer pair, PCR was performedwith human genome (CLONTECH) as a template. An amplified fragment ofapprox. 0.24 kb was isolated, and after digestion with restrictionenzymes XhoI and HindIII, the fragment was inserted between XhoI andHindIII sites of plasmid pU1 prepared above using T4 DNA ligase.Thereafter, E. coli DH5 (TOYOBO) was transformed with the plasmid toprepare an expression vector pUH1.

[0363] In the above, PCR was performed by preparing 50 μl of reactionsolution comprising 1 μl of KOD-Plus-(TOYOBO Co. Ltd.), 5 μl of 10×PCRBuffer (supplied with KOD-Plus-), 5 μl of 2 mM dNTPs (supplied withKOD-Plus-), 2 μl of 25 mM MgSO₄ (supplied with KOD-Plus-), 1.5 μl ofeach of the above primers (each at a concentration of 10 μM) and 100 ngof human Genomic DNA (CLONTECH), and after 2 minutes incubation at 94°C., conducting 45 cycles of incubation for 15 seconds at 94° C., 30seconds at 60° C., and 40 seconds at 68° C. using Takara PCR ThermalCycler MP (manufactured by Takara Bio Inc.), to thereby obtain eachfragment.

[0364] (2) Preparation of siRNA Expression Vectors Targeting the Gene ofSEQ ID NO: 88

[0365] The synthetic oligonucleotides (DNA) of SEQ ID NO: 197 and SEQ IDNO: 198 were dissolved to a final concentration of 10 μM in an annealingbuffer (100 mM potassium acetate, 30 mM HEPES-KOH pH 7.4, 2 mM magnesiumacetate), and after heating the solution for 4 minutes at 94° C., thesolution was incubated for 1 hour at 37° C. to effect annealing. Theobtained double stranded oligonucleotide was inserted between BbsI andHindIII sites of the expression vector pUH1 using T4 DNA ligase.Thereafter, E. coli DH5 (TOYOBO) was transformed with the resultantvector, and the siRNA expression vector was purified using QIAfilterPlasmid Kit (QIAGEN). The siRNA expression vector was given thedesignation pUH88-1.

[0366] siRNA expression vector pUH88-2 was prepared from the syntheticoligonucleotides of SEQ ID NO: 199 and SEQ ID NO: 200, by the samemethod as described above.

[0367] siRNA expression vector pUH88-3 was prepared from the syntheticoligonucleotides of SEQ ID NO: 201 and SEQ ID NO: 202, by the samemethod as described above.

[0368] siRNA expression vector pUH88-4 was prepared from the syntheticoligonucleotides of SEQ ID NO: 203 and SEQ ID NO: 204, by the samemethod as described above.

[0369] siRNA expression vector pUH88-5 was prepared from the syntheticoligonucleotides of SEQ ID NO: 205 and SEQ ID NO: 206, by the samemethod as described above.

[0370] siRNA expression vector pUH88-6 was prepared from the syntheticoligonucleotides of SEQ ID NO: 207 and SEQ ID NO: 208, by the samemethod as described above.

[0371] (3) Expression Inhibition of the Gene of SEQ ID NO: 88 by siRNAExpression vector prepared in (2) above.

[0372] EBNA cells (Invitrogen) were inoculated on DMEM medium containing5% FBS in a 96-well cell culture plate to a cell density of 1.2×10⁴cells/100 μl/well, and cultured for 24 hours at 37° C. in the presenceof 5% CO₂. Next, using FuGENE6, 50 ng of an expression vector having thepolynucleotide of SEQ ID NO:88 and 50 ng of pNFkB-Luc (STRATAGENE) and10 ng of phRL-TK vector (Promega Corporation) used as an internalstandard, were co-transfected with the siRNA expression vector pUH88-1(50 ng) prepared in the above (2) into 1 well. After culturing for 24hours at 37° C., firefly luciferase activity and Renilla reniformisluciferase activity were measured using Dual-Luciferase Reporter AssaySystem (Promega Corporation). Further, each of pUH88-2 (50 ng), pUH88-3(50ng), pUH88-4 (50 ng), pUH88-5 (50 ng) or pUH88-6 (50 ng) wasco-transfected into 1 well in place of pUH88-1 (50 ng), and luciferaseactivity was measured in the same manner. Results are shown in Table 2.In Table 2, values for activity represent relative values when fireflyluciferase activity in the control experiment (cells into which pUH1 wasintroduced) is established as 100%. (It should be noted that values forfirefly luciferase activity were standardized with values for Renillareniformis luciferase activity which was used as an internal standard,before using these values in calculations.) As indicated in Table 2,cells into which expression vector pUH88-1, pUH88-2, pUH88-3, pUH88-4,pUH88-5 or pUH88-6 is introduced, exhibited inhibition o fireflyluciferase activity relative to the control experiment (cell into whichpUH-1 was introduced). These results indicated that siRNA expressed bypUH88-1, pUH88-2, pUH88-3, pUH88-4, pUH88-5 and pUH88-6 inhibitedexpression of the gene of SEQ ID NO:88, respectively. TABLE 2 VectorRelative Luciferase Activity (%) pUH1 100 pUH88-1 20 pUH88-2 10 pUH88-38 pUH88-4 7 pUH88-5 4 pUH88-6 6

INDUSTRIAL APPLICABILITY

[0373] As described above, the present invention provides industriallyhighly useful proteins capable of activating NF-κ B and genes encodingthe proteins. The proteins of the present invention and the genesencoding the proteins allow not only screening for compounds useful fortreating and preventing diseases associated with the excessiveactivation or inhibition of NF-κ B, but also production of diagnosticsfor such diseases. The genes of the present invention are also useful asa gene source used for gene therapy.

[0374] All publications, patents and patent applications cited hereinare incorporated herein in their entirety.

0 SEQUENCE LISTING The patent application contains a lengthy “SequenceListing” section. A copy of the “Sequence Listing” is available inelectronic form from the USPTO web site(http://seqdata.uspto.gov/sequence.html?DocID=20040081986). Anelectronic copy of the “Sequence Listing” will also be available fromthe USPTO upon request and payment of the fee set forth in 37 CFR1.19(b)(3).

1. A purified protein selected from the group consisting of: (a) aprotein which consists of an amino acid sequence represented by any oneof SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29,31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65,67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101,103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129,131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 152, 154, 156, 158,160, 162, 164, 166, 168, 170, 172, 174, 176, 178 and 180; and (b) aprotein that activates NF-κ B (Nuclear factor kappa B) and consists ofan amino acid sequence having at least one amino acid deletion,substitution or addition in an amino acid sequence represented by anyone of SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27,29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63,65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99,101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127,129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 152, 154, 156,158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178 and 180:
 2. Apurified protein that activates NF-κ B and comprises an amino acidsequence having at least 95% identity to the protein according to claim1 over the entire length thereof.
 3. An isolated polynucleotide whichcomprises a nucleotide sequence encoding a protein selected from thegroup consisting of: (a) a protein which comprises an amino acidsequence represented by any one of SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13,15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49,51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85,87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117,119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145,147, 149, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174,176, 178 and 180; and (b) a protein that activates NF-κ B and consistsof an amino acid sequence having at least one amino acid deletion,substitution or addition in an amino acid sequence represented by anyone of SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27,29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63,65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99,101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127,129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 152, 154, 156,158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178 and
 180. 4. Anisolated polynucleotide comprising a polynucleotide sequence selectedfrom the group consisting of: (a) a polynucleotide sequence representedby any one of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24,26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60,62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96,98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124,126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 151,153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173, 175, 177 and 179;(b) a polynucleotide sequence encoding a protein that activates NF-κ Band hybridizing under stringent conditions with a polynucleotide havinga polynucleotide sequence complementary to the polynucleotide sequenceof (a); and (c) a polynucleotide sequence which encodes a protein thatactivates NF-κ B and consists of a polynucleotide sequence having atleast one nucleotide deletion, substitution or addition in apolynucleotide sequence represented by any one of SEQ ID NOS: 2, 4, 6,8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42,44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78,80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110,112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138,140, 142, 144, 146, 148, 150, 151, 153, 155, 157, 159, 161, 163, 165,167, 169, 171, 173, 175, 177 and
 179. 5. An isolated polynucleotidecomprising a polynucleotide sequence selected from the group consistingof: (a) a nucleotide sequence represented by a coding region in any oneof SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30,32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66,68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100,102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128,130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 151, 153, 155,157, 159, 161, 163, 165, 167, 169, 171, 173, 175, 177 and 179; (b) anucleotide sequence encoding a protein that activates NF-κ B andhybridizing under stringent conditions with a polynucleotide having apolynucleotide sequence complementary to the polynucleotide sequence of(a); and (c) a nucleotide sequence which encodes a protein thatactivates NF-κ B and consists of a nucleotide sequence having at leastone nucleotide deletion, substitution or addition in a nucleotidesequence represented by a coding region in any one of SEQ ID NOS: 2, 4,6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40,42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76,78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108,110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136,138, 140, 142, 144, 146, 148, 150, 151, 153, 155, 157, 159, 161, 163,165, 167, 169, 171, 173, 175, 177 and
 179. 6. An isolated polynucleotidecomprising a nucleotide sequence which encodes a protein that activatesNF-κ B and has at least 95% identity to the polynucleotide sequenceaccording to claim 3 over the entire length thereof.
 7. An isolatedpolynucleotide comprising a nucleotide sequence which encodes a proteinthat activates NF-κ B and has at least 95% identity to thepolynucleotide sequence according to claim 4 or 5 over the entire lengththereof.
 8. A purified protein encoded by the polynucleotide accordingto any one of claims 3 to
 7. 9. A recombinant vector which comprises apolynucleotide according to any one of claims 3 to
 7. 10. A gene thrapyagent comprising the recombinant vector according to claim 9 as anactive ingredient.
 11. A transformed cell which comprises therecombinant vector according to claim
 9. 12. A membrane of the cellaccording to claim 11, when the protein according to claim 1 or 2 is amembrane protein.
 13. A process for producing a protein comprising, (a)culturing a transformed cell comprising the isolated polynucleotideaccording to any one of claims 3 to 7 under conditions providingexpression of the encoded protein; and (b) recovering the protein fromthe culture product.
 14. A process for diagnosing a disease orsusceptibility to a disease related to expression or activity of theprotein of claim 1, 2 or 8 in a subject comprising: (a) determining thepresence or absence of a mutation in the nucleotide sequence encodingsaid protein in the genome of said subject; and/or (b) analyzing theamount of expression of said protein in a sample derived from saidsubject.
 15. A method for screening compounds in respect of activity toinhibit or promote NF-κ B activation, which comprises the steps of: (a)providing a cell with a gene encoding a protein that activates NF-κ B,and a component that provides a detectable signal associated withactivation of NF-κ B; (b) culturing a transformed cell under conditions,which permit the expression of the gene in the transformed cell; (c)contacting the transformed cell with one or more compounds; (d)measuring the detectable signal; and (e) isolating or identifying anactivator compound and/or an inhibitor compound by measuring thedetectable signal.
 16. A process for producing a pharmaceuticalcomposition, which comprises the steps of: (a) providing a cell with agene encoding a protein that activates NF-κ B, and a component capableof providing a detectable signal; (b) culturing a transformed cell underconditions, which permit the expression of the gene in the transformedcell; (c) contacting the transformed cell with one or more compounds;(d) measuring the detectable signal; (e) isolating or identifying anactivator compound and/or an inhibitor compound by measuring thedetectable signal; and (f) optimizing the isolated or identifiedcompound as a pharmaceutical composition.
 17. A kit for screening acompound in respect of activity to inhibit or promote NF-κ B activation,which comprises: (a) a cell comprising a gene encoding a protein thatactivates NF-κ B, and a component that provides a detectable signal uponactivation of NF-κ B; and (b) reagents for measuring the detectablesignal.
 18. A monoclonal or polyclonal antibody or a fragment thereofthat specifically binds to the protein according to claim 1, 2 or
 8. 19.The monoclonal or polyclonal antibody or a fragment thereof according toclaim 18 that inhibits the action of activating NF-κ B of the proteinaccording to claim 1, 2 or
 8. 20. A process for producing a monoclonalor polyclonal antibody that specifically binds to the protein accordingto claim 1, 2 or 8, which comprises administering the protein accordingto claim 1, 2 or 8 or epitope-bearing fragments thereof to a non-humananimal.
 21. An antisense oligonucleotide complementary to thepolynucleotide according to any one of claims 3 to 7, which preventsNF-κ B activator protein expression.
 22. A ribozyme which inhibits NF-κB activation by cleavage of RNA that encodes the protein according toclaim 1, 2 or
 8. 23. A double stranded nucleic acid having a nucleotidesequence corresponding to a part of the nucleotide sequence of theisolated polynucleotide according to any one of claims 3-7, whichinhibit the expression of the protein that activates NF-κ B.
 24. Thedouble stranded nucleic acid according to claim 23, wherein the nucleicacid has a nucleotide sequence corresponding to a part of the nucleotidesequence represented by SEQ ID NO: 88, and inhibits the expression ofthe protein having the amino acid sequence represented by SEQ ID NO: 87.25. A double stranded nucleic acid obtained by annealing of any one ofthe following oligonucleotide pairs (a)-(f): (a)5′-GUCCAGGAUAUCAUGAGUCN_(n)-3′ (SEQ ID NO:213)3′-N_(n)CAGGUCCUAUAGUACUCAG-5′ (SEQ ID NO:214) (b)5′-GAAGUCUGAAGAUCUAUCCN_(n)-3′ (SEQ ID NO:215)3′-N_(n)CUUCAGACUUCUAGAUAGG-5′ (SEQ ID NO:216) (c)5′-GCUGAAGAAGAGGUGUUCCN_(n)-3′ (SEQ ID NO:217)3′-N_(n)CGACUUCUUCUCCACAAGG-5′ (SEQ ID NO:218) (d)5′-GAUGACACAGAUGAAGCCCN_(n)-3′ (SEQ ID NO:219)3′-N_(n)CUACUGUGUCUACUUCGGG-5′ (SEQ ID NO:220) (e)5′-GCCCUCAGAGUCCAGAAUCN_(n)-3′ (SEQ ID NO:221)3′-N_(n)CGGGAGUCUCAGGUCUUAG-5′ (SEQ ID NO:222) (f)5′-GAUGACUUUGGUAUCAAACN_(n)-3′ (SEQ ID NO:223)3′-N_(n)CUACUGAAACCAUAGUUUG-5′ (SEQ ID NO:224)

wherein N represents any one of G, A, T, C, and U, and n is 1 to
 4. 26.The double stranded nucleic acid according to claim 25, wherein Nn is TTor UU.
 27. A double stranded nucleic acid having one or more mutationsin the sense strand of the double strand nucleic acid according to claim26.
 28. A double stranded nucleic acid comprising the double strandednucleic acid according to any one of claims 25 to 27 as a part, whichinhibits the expression of the protein having the amino acid sequencerepresented by SEQ ID NO:
 87. 29. An expression vector capable ofexpressing the double stranded nucleic acid according to claim 25,wherein Nn is UU or UUU.
 30. A method for treating a disease, whichcomprises administering to a subject an amount of compound screened bythe process according to claim 15, and/or a monoclonal or polyclonalantibody or a fragment thereof according to claim 18 or 19, and/or anantisense oligonucleotide according to claim 21 and/or a ribozymeaccording to claim 22 and/or a double stranded nucleic acid according toany one of claims 23-28 and/or the expression vector according to claim29 effective to treat a disease selected from the group consisting ofinflammation, autoimmune diseases, infectious disease, cancers and bonediseases.
 31. A method for treating a disease, which comprisesadministering to a subject an amount of a compound screened by theprocess according to claim 15, and/or a monoclonal or polyclonalantibody or a fragment thereof according to claim 18 or 19, and/or anantisense oligonucleotide according to claim 21 and/or a ribozymeaccording to claim 22 effective to treat a disease selected from thegroup consisting of inflammation, autoimmune diseases, infectiousdisease, cancers and bone diseases.
 32. A pharmaceutical compositionproduced by the process according to claim 16 as an inhibitor orpromoter of NF-κ B activation.
 33. A pharmaceutical compositionaccording to claim 32 for the treatment of inflammation, autoimmunediseases, cancers, infectious diseases, bone diseases, AIDS,neurodegenerative diseases or ischemic disorders.
 34. A method oftreating inflammation, autoimmune diseases, cancers, infectiousdiseases, bone diseases, AIDS, neurodegenerative diseases, or ischemicdisorders, which comprises administering a pharmaceutical compositionproduced by the process according to claim 16 to a patient sufferingfrom a disease associated with NF-κ B activation.
 35. A pharmaceuticalcomposition which comprises a monoclonal or polyclonal antibody or afragment thereof according to claim 18 or 19 as an active ingredient.36. A pharmaceutical composition which comprises an antisenseoligonucleotide according to claim 21 as an active ingredient.
 37. Apharmaceutical composition which comprises a ribozyme according to claim22 as an active ingredient.
 38. A pharmaceutical composition or a genetherapy agent which comprises a double stranded nucleic acid accordingto any one of claims 23 to 28 and/or an expression vextor according toclaim 29 as an active ingredient.
 39. An expression inhibiting agent fora protein having an action of activating NF-κ B, which comprises adouble stranded nucleic acid according to any one of claims 23 to 28and/or an expression vextor according to claim 29 as an activeingredient.
 40. The pharmaceutical composition according to claim 35 or36, wherein the target disease is selected from the group consisting ofinflammation, autoimmune diseases, infectious diseases, cancers, bonediseases, AIDS, neurodegenerative and ischemic disorders.
 41. A methodfor obtaining a novel gene having a function, which comprises at leastthe following steps: (a) constructing a full-length cDNA library by theoligo-capping method; (b) cotransfecting the full-length cDNA and aplasmid containing a factor emitting a signal indicative of the presenceof a protein having the function into cells; and (c) selecting a plasmidemitting the signal.
 42. A computer-readable medium on which a sequencedata set has been stored, said sequence data set comprising at least onenucleotide sequence selected from the group consisting of SEQ ID NOS: 2,4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40,42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76,78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108,110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136,138, 140, 142, 144, 146, 148, 150, 151, 153, 155, 157,159, 161, 163,165, 167, 169, 171, 173, 175, 177 and 179, and/or at least one aminoacid sequence selected from the group consisting of SEQ ID NOS: 1, 3, 5,7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41,43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77,79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109,111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137,139, 141, 143, 145, 147, 149, 152, 154, 156, 158, 160, 162, 164, 166,168, 170, 172, 174, 176, 178 and
 180. 43. A method for calculatingidentity to other nucleotide sequences and/or amino acid sequences,which comprises comparing data on a medium according to claim 42 withdata of said other nucleotide sequences and/or amino acid sequences. 44.An insoluble substrate to which polynucleotides comprising all or partof the nucleotide sequences selected from the group consisting of SEQ IDNOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36,38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72,74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106,108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134,136, 138, 140, 142, 144, 146, 148, 150, 151, 153, 155, 157, 159, 161,163, 165, 167, 169, 171, 173, 175, 177 and 179, are fixed.
 45. Aninsoluble substrate to which polypeptides comprising all or a part ofthe amino acid sequences selected from the group consisting of SEQ IDNOS: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35,37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71,73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105,107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133,135, 137, 139, 141, 143, 145, 147, 149, 152, 154, 156, 158, 160, 162,164, 166, 168, 170, 172, 174, 176, 178 and 180, are fixed.